Patent Application
20100107265
This invention relates generally to factors affecting muscle development in mammals, especially livestock. In particular, this invention relates to the cloning of the myostatin gene, a member of the TGF-β superfamily, its involvement in muscular hyperplasia in livestock, and a method for determining myostatin genotypes. This invention most particularly relates to the production of transgenic non-human male mammals exhibiting muscular hypertrophy, such as transgenic bovine.
The TGF-β superfamily consists of a group of multifunctional polypeptides which control a wide range of differentiation processes in many mammalian cell types. GDF-8 is a member of the TGF-β superfamily. All members of this superfamily share a common structure including a short peptide signal for secretion and an N-terminal peptide fragment that is separated from the bioactive carboxy-terminal fragment by proteolytic cleavage at a highly conserved proteolytic cleavage site. The bioactive carboxy-terminal domain is characterized by cysteine residues at highly conserved positions which are involved in intra- and intermolecular disulfide bridges. The functional molecules are covalently linked (via a S—S bond) dimers of the carboxy-terminal domain (U.S. Pat. No. 5,827,733).
Recently, it was reported that mice deficient in the gene encoding for GDF-8 were characterized by a generalized muscular hyperplasia (McPherron et al. Nature 387:83-90 1997). The GDF-8 deficient mice were produced by gene targeting using homologous recombination in embryonic stem cells, a method referred to as “gene knock-out”. The murine generalized muscular hyperplasia appeared to be very similar in its expression to the muscular hyperplasia characterizing “double-muscled” cattle. This observation raised the intriguing possibility that the bovine gene encoding for GDF-8(i.e. the bovine evolutionary homologue of the mouse GDF-8 gene) is involved in the bovine double-muscling phenotype. It also raised the possibility that the human gene coding for GDF-8 (i.e. the human evolutionary homologue of the mouse GDF-8 gene) is involved in regulating muscular development in humans, specifically skeletal muscle genesis. Isolation of the human GDF-8 gene may have therapeutic uses/applications in the treatment of musculodegenerative diseases through upgrading or downgrading the expression of GDF-8.
The occurrence of animals characterized by a distinct generalized muscular hypertrophy, commonly known as “double-muscled” animals, has been reported in several cattle breeds throughout the world. The first documented description of double-muscled cattle dates back as early as 1807 (Culley, G Observations on Livestock, 4th edition, London, G. Woodfall, 1807). One of the breeds in which this characteristic has been most throughly analyzed is the Belgian Blue Cattle Breed (“Belgian Blue Breed”). This is one of the only breeds where the double-muscled trait has been systematically selected for, and where the double-muscled phenotype is virtually fixed. A comparison of double-muscled and conventional animals within the Belgian Blue Breed, showed an increase in muscle mass by 20% on average, while all other organs are reduced in size (Hanset, R. In Breeding for Disease Resistance in Farm Animals, Owen, Axford, editor, C.A.B. International, pages 467-478 1991). The muscular hypertrophy was shown to be an histological hyperplasia affecting primarily superficial muscles, accompanied by a 50% reduction in total lipid content and a reduction in connective tissue fraction as measured by hydroxyproline content (Hanset et al. In Current Topics in Veterinary Medicine and Animal Science, volume 16:341-349 Eds. King and Mènissier 1982). Double-muscled animals were shown to have a reduced feed intake with improved feed conversion ratio (Hanset et al. Gènèt. Sèl. Evol. 19:225-248 1987). An important economic benefit of double-muscled animals, in contrast to conventional animals, is the substantial increase in selling price and net income for the farmer (Hanset et al. 1987).
One of the most through series of studies on double-muscling is that of Hanset and colleagues in the Belgian Blue Breed. Objective criteria of muscular development, such as dressing-out percentage, lean and fat percentage, plasma and red cell creatine and creatinine concentrations, were measured on nearly 150 randomly selected animals raised in standardized conditions. These studies clearly revealed abnormal, bimodal distributions of the double-muscled phenotype and objectively confirmed the visual classification traditionally performed by breeders on double-muscled and conventional animals. The phenotypic distribution was resolved using a maximum likelihood procedure into two component normal populations with a common variance which revealed mean differences of three to four standard deviations depending on the trait. This suggested the presence of an allele having a major effect on muscular development with a population frequency close to 50% (Hanset and Michaux Gènèt. Sèl. Evol. 17:369-386 1985). The most convincing evidence in favor of such an allele, however, came from experimental crosses involving double-muscled Belgian Blue sires and Holstein Friesian dairy cows (the latter animals having very poor muscular development). While F1 offspring showed a phenotypic distribution very similar to that of Holstein Friesian dams, backcrossing these F1′s to double-muscled sires produced a bimodal BC generation, clearly pointing towards the Mendelian segregation of a recessive “mh” (muscular hypertrophy) allele (Hanset and Michaux Gènèt. Sèl. Evol. 17:359-368 1985).
The same kind of experimental crosses were subsequently used to perform a whole genome scan using a microsatellite based marker map. To perform the linkage analysis, animals were classified as double-muscled or conventional. Very significant Logarithm of the Odds scores (lodscores) were obtained on chromosome 2 (>17), and multi-point linkage analysis positioned on the mh locus at the centromeric end of this chromosome, at [2]centimorgan from the nearest microsatellite marker: TGLA44. The corresponding chromosomal region accounted for all of the variance of the trait assumed to be fully penetrant in this experiment (Charlier et al. Mammalian Genome 6:788-792 1995).
Intensive breeding programs implemented over the last 50 years have created cattle breeds that are highly specialized in either milk production (e.g. Holstein-Friesian and Jersey) or meat production (e.g. Angus, Hereford, Charolais, Piedmontese, and Belgian Blue). Physiological antagonisms have indeed precluded combining superior abilities for both milk and meat production in the same animal. Despite its effectiveness, the resulting production system can be considered suboptimal because of poor carcass and milk yield of beef and diary cattle, respectively. Thus, the art lacks a production system that efficiently increases both milk yield and carcass yield in the same cattle population.
Furthermore, in humans, genes coding for some forms of muscular abnormalities have been isolated, e.g. muscular dystrophy. The present invention provides for the gene which regulates the development of skeletal muscle only, as opposed to other types of muscle, e.g. smooth or cardiac muscle. The present invention may provide an understanding of the role of the GDF-8 gene or its receptor in the regrowth of skeletal muscle in humans which only undergoes a hyperplasic response. The transgenic animals provided by the instant invention can be used as research tools to increase the understanding of the pathogenesis of disease in the muscular-skeletal system and to aid in the development of means to diagnosis and/or treat such diseases.
The present inventors have identified and sequenced a gene (cDNA and genomic) encoding a bovine myostatin protein. The nucleic acid coding sequence is identified as SEQ ID NO:1 and the protein sequence is identified as SEQ ID NO:2. The genomic bovine sequence is identified as SEQ ID NO:54. A mutant gene (SEQ ID NO:3) in which the coding sequence lacks an 11-base pair consecutive sequence (SEQ ID NO:11) of the sequence encoding bovine protein having myostatin activity has been sequenced. It has been shown that cattle of the Belgian Blue breed homozygous for the mutant gene lacking myostatin activity are double-muscled. Other bovine mutations which lead to double-muscling have also been determined, being identified herein as nt419(de17-ins10), Q204X, E226X and C313Y, respectively.
In one aspect, the present invention thus provides a method for determining the presence of muscular hyperplasia in a mammal. The method includes obtaining a sample of material containing DNA from the mammal and ascertaining whether a sequence of the DNA encoding (a) a protein having the biologicalactivity of myostatin, is present, and whether a sequence encoding of the DNA encoding (b) an allelic protein lacking the activity of (a), is present. The absence of (a) and the presence of (b) indicates the presence of muscular hyperplasia in the mammal.
Of course, the mutation responsible for the lack of activity can be a naturally occurring mutation, as in the case for the Belgian Blue, Asturiana, Parthenaise or Rubia Gallega breeds, shown here.
The mammals of the instant invention are preferably, but not limited to, cattle.
There are several methods known for determining whether a particular nucleotide sequence is present in a sample. A common method is the polymerase chain reaction (PCR). A preferred aspect of the invention thus includes a step in which ascertaining whether a sequence of the DNA encoding (a) is present, and whether a sequence of the DNA encoding (b) is present includes amplifying the DNA in the presence of primers based on a nucleotide sequence encoding a protein having the biological activity of myostatin.
A primer of the present invention, used in PCR for example, is a nucleic acid molecule sufficiently complementary to the sequence on which it is based and of sufficient length to selectively hybridize to the corresponding portion of a nucleic acid molecule intended to be amplified and to prime synthesis thereof under in vitro conditions commonly used in PCR. Likewise, a probe of the present invention, is a molecule, for example a nucleic acid molecule of sufficient length and sufficiently complementary to the nucleic acid molecule of interest, which selectively binds under high or low stringency conditions with the nucleic acid sequence of interest for detection thereof in the presence of nucleic acid molecules having differing sequences.
In preferred aspects, primers are based on the sequences identified as SEQ ID NO:7 or SEQ ID NO:54.
In another aspect, the invention is a method for determining the presence of muscular hyperplasia in a mammal which includes obtaining a sample of material containing mRNA from the mammal. Such method includes ascertaining whether a sequence of the mRNA encoding (A) a protein having the biologicalactivity of myostatin, is present, and whether a sequence of the mRNA encoding (B) a protein at least partially encoded by a truncated nucleotide sequence corresponding to substantially the sequence of the mRNA and lacking the activity of (A), is present. The absence of (A) and the presence of (B) indicates the presence of muscular hyperplasia in the mammal.
The mRNA encoding (A) and the truncated sequence can correspond to alleles of DNA of the mammal.
Again, if an amplification method such as PCR is used in ascertaining whether a sequence of the mRNA encoding (A) is present, and whether a sequence of the mRNA encoding (B) is present, the method includes amplifying the mRNA in the presence of a pair of primers complementary to a nucleotide sequence encoding a protein having the biological activity of myostatin. Each such primer can contain a nucleotide sequence substantially complementary, for example, to the sequence identified as SEQ ID NO:7. The truncated sequence can contain at least 50 consecutive nucleotides substantially corresponding to 50 consecutive nucleotides of SEQ ID NO:7, for example.
In another aspect, the invention is a method for determining the presence of muscular hyperplasia in a mammal which includes obtaining a tissue sample containing mRNA of the mammal and ascertaining whether an mRNA encoding a mutant type myostatin protein lacking the biologicalactivity of myostatin is present. The presence of such an mRNA encoding a mutant type myostatin protein indicates the presence of muscular hyperplasia in the mammal.
In another aspect, the invention thus provides a method for determining the presence of muscular hyperplasia in a bovine animal. The method includes obtaining a sample of material containing DNA from the animal and ascertaining whether DNA having a nucleotide sequence encoding a protein having the biological activity of myostatin is present. The absence of DNA having such a nucleotide sequence indicates the presence of muscular hyperplasia in the animal. Ascertaining whether DNA having a nucleotide sequence encoding a protein having the biological activity of myostatin can include amplifying the DNA in the presence of primers based on a nucleotide sequence encoding a protein having the biological activity of myostatin.
In particular, the method can be carried out using a sample from an animal in which such a bovine animal not displaying muscular hyperplasia is known to have a nucleotide sequence which is capable of hybridizing with a nucleic acid molecule having the sequence identified as SEQ ID NO:1 under stringent hybridization conditions.
It is possible that ascertaining whether DNA having a nucleotide sequence encoding a protein having the biologicalactivity of myostatin is present includes amplifying the DNA in the presence of primers based on a nucleotide sequence encoding the N-terminal and the C-terminal, respectively, of the protein having the biological activity of myostatin.
Primers, say first and second primers, can be based on first and second nucleotide sequences encoding spaced apart regions of the protein, wherein the regions flank a mutation known to naturally occur and which when present in both alleles of such an animal results in muscular hyperplasia.
It can also be that DNA of such an animal not displaying muscular hyperplasia contains a nucleotide sequence which hybridizes under stringent conditions with a nucleotide sequence encoding a protein having a sequence identified as SEQ ID NO:2 and the coding sequence of DNA of such an animal displaying muscular hyperplasia is known to contain an 11-base pair deletion beginning at base pair number 821 of the coding sequence, and said first primer is selected to be upstream of the codon encoding glutamic acid number 275 and the second primer is selected to be downstream of the codon encoding aspartic acid number 274.
Also, a DNA of such an animal not displaying muscular hyperplasia might contain a nucleotide sequence which hybridizes under stringent conditions with a nucleotide sequence encoding a protein having a sequence identified as SEQ ID NO:2. The coding sequence of DNA of such an animal displaying muscular hyperplasia might be known to contain an 11-base pair deletion beginning at base pair number 821. A primer can be selected to span the nucleotide sequence including base pair numbers 820 and 821 of the DNA sequence containing the deletion.
The animal can be of the Belgian Blue breed.
In a particular aspect, ascertaining whether DNA having a nucleotide sequence encoding a protein having the biological activity of myostatin is present includes amplifying the DNA in the presence of a primer containing at least a portion of a mutation known to naturally occur and which when present in both alleles of a said animal results in muscular hyperplasia.
In another aspect, the invention is a method for determining the presence of muscular hyperplasia in a bovine animal which includes obtaining a sample of the animal containing mRNA and ascertaining whether an mRNA encoding a protein having the biological activity of myostatin is present in the sample. The absence of said mRNA indicates the presence of said muscular hyperplasia in the animal.
A sample containing mRNA is preferably, but not limited to, skeletal muscle tissue.
In a particular aspect, the invention is a method for determining the presence of double-muscling in a bovine animal, involving obtaining a sample of material containing DNA from the animal and ascertaining whether the DNA contains the nucleotide sequence identified as SEQ ID NO:11 in which the absence of the sequence indicates double-muscling in the animal.
The animal is preferably of, but not limited to, the Belgian Blue breed.
In another aspect, the invention is a method for determining the myostatin genotype of a mammal, as may be desirable to know for breeding purposes. The method includes obtaining a sample of material containing nucleic acid of the mammal, wherein the nucleic acid is uncontaminated by heterologous nucleic acid; ascertaining whether the sample contains a(i) nucleic acid molecule encoding a protein having the biological activity of myostatin; and ascertaining whether the sample contains an (ii) allelic nucleic acid molecule encoding a protein lacking the biological activity of myostatin. The mammal can be bovine.
In another aspect, the subject is human and (i) includes a nucleic acid sequence substantially homologous (in the sense of identity) with the sequence identified as SEQ ID NO:7.
The invention includes a method of increasing muscle mass of a mammal having muscle cells in which myostatin is expressed, the method comprising administering to the mammal an effective amount of a nucleic acid molecule substantially complementary to at least a portion of mRNA encoding myostatin and being of sufficient length to sufficiently reduce expression of the myostatin to increase the muscle mass. In a particularly preferred aspect, the mammal is bovine.
In another embodiment, the invention is a method of increasing muscle mass of a mammal, including administering to the mammal an effective amount of a nucleic acid molecule having ribozyme activity and a nucleotide sequence substantially complementary to at least a portion of mRNA encoding myostatin and being of sufficient length to bind selectively thereto to sufficiently reduce expression of the myostatin so as to increase the muscle mass.
The invention includes a diagnostic kit, for determining the presence of muscular hyperplasia in a mammal from which a sample containing DNA of the mammal has been obtained. The kit includes first and second primers for amplifying the DNA, the primers being complementary to nucleotide sequences of the DNA upstream and downstream, respectively, of a mutation in the portion of the DNA encoding myostatin which results in muscular hyperplasia of the mammal, wherein at least one of the nucleotide sequences is selected to be from a non-coding region of the myostatin gene. This kit can also include a third primer complementary to a naturally occurring mutation of a coding portion of the myostatin gene.
A particular diagnostic kit, for determining the genotype of a sample of mammalian genetic material, particularly bovine material, includes a pair of primers for amplifying a portion of the genetic material corresponding to a nucleotide sequence which encodes at least a portion of a myostatin protein, wherein a first of the primers includes a nucleotide sequence sufficiently complementary to a mutation of SEQ ID NO:1 to prime amplification of a nucleic acid molecule containing the mutation, the mutation being selected from a group of mutations resulting from: (a) deletion of 11 nucleotides beginning at nucleotide 821 of the coding portion of SEQ ID NO:1; (b) deletion of 7 nucleotides beginning at nucleotide 419 of the coding sequence and insertion of the sequence AAGCATACAA (SEQ ID NO:55) in place thereof; (c) deletion of nucleotide 204 of the coding sequence and insertion of T in place thereof; (d) deletion of nucleotide 226 of the coding sequence and insertion of T in place thereof; and (e) deletion of nucleotide 313 of the coding sequence and insertion of A in place thereof; and combinations thereof. The second of the pair of primers is preferably located entirely upstream or entirely downstream of the selected mutation or mutations. In one kit, a first said primer spans mutation (a) and further comprising a third primer which is sufficiently complementary to the nucleotide sequence identified as SEQ ID NO:11 to prime amplification of a nucleic acid molecule containing SEQ ID NO:11. In another (or the same kit), a first said primer is sufficiently complementary to the inserted sequence of mutation (b) to prime amplification of a nucleic acid molecule containing mutation (b) and further comprising a third primer which is sufficiently complementary to the sequence corresponding to the 7 nucleotide deletion of mutation (b) to prime amplification of a nucleic acid molecule containing the 7 nucleotide deletion of mutation (b). In another (or the same kit), a first said primer spans mutation (c) and further comprising a third primer which is sufficiently complementary to the sequence spanning the corresponding region lacking the mutation (c) to prime amplification of a nucleic acid molecule lacking mutation (c). In another (or the same kit), a first said primer spans mutation (d) and further comprising a third primer which is sufficiently complementary to the sequence spanning the corresponding region lacking mutation (d) to prime amplification of a nucleic acid molecule lacking mutation (d). In another (or the same kit), a first said primer spans mutation (e) and further comprising a third primer which is sufficiently complementary to a sequence spanning the corresponding region lacking mutation (e) to prime amplification of a nucleic acid molecule lacking mutation (e).
The invention includes a purified protein having the biological activity of myostatin, and having an amino acid sequence identified as SEQ ID NO:2, or a conservatively substituted variant thereof. The invention includes a purified bovine protein having the biological activity of myostatin or a purified human protein having the biologicalactivity of myostatin.
The invention includes an isolated nucleic acid molecule encoding a foregoing protein. Particularly, the invention includes an isolated nucleic acid molecule comprising a DNA molecule having the nucleotide sequence identified as SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:7 or which varies from the sequence due to the degeneracy of the genetic code, or a nucleic acid strand capable of hybridizing with at least one said nucleic acid molecule under stringent hybridization conditions.
The invention includes isolated mRNA transcribed from DNA having a sequence which corresponds to a nucleic acid molecule of the invention.
The invention includes an isolated DNA in a recombinant cloning vector and a microbial cell containing and expressing heterologous DNA of the invention.
The invention includes a transfected cell line which expresses a protein of the invention.
The invention includes a process for producing a protein of the invention, including preparing a DNA fragment including a nucleotide sequence which encodes the protein; incorporating the DNA fragment into an expression vector to obtain a recombinant DNA molecule which includes the DNA fragment and is capable of undergoing replication; transforming a host cell with recombinant DNA molecule to produce a transformant which can express the protein; culturing the transformant to produce the protein; and recovering the protein from resulting cultured mixture.
The invention includes a method of inhibiting myostatin so as to induce increased muscle mass in a mammal, comprising administering an effective amount of an antibody to myostatin to the mammal.
The invention includes a method of increasing muscle mass in a mammal, by raising an autoantibody to the myostatin in the mammal. Raising the autoantibody can include administering a protein having myostatin activity to the mammal.
The invention includes a method of increasing muscle mass in a mammal including administering to the mammal an effective amount of an antisense nucleic acid or oligonucleotide substantially complementary to at least a portion of the sequence identified as SEQ ID NO:1 or SEQ ID NO:5, or SEQ ID NO:7. The portion can be at least 5 nucleotide bases in length or longer. The mammal can be a bovine and the sequence can be that identified as SEQ ID NO:l.
The invention includes a method of inhibiting production of myostatin in a mammal in need thereof, including administering to the mammal an effective amount of an antibody to the myostatin.
The invention includes a probe containing a nucleic acid molecule sufficiently complementary with a sequence identified as SEQ ID NO:1, or its complement, so as to bind thereto under stringent conditions. The probe can be a sequence which is between about 8 and about 1195 nucleotides in length.
The invention includes a primer composition useful for detection of the presence of DNA encoding myostatin in cattle. The composition can include a nucleic acid primer substantially complementary to a nucleic acid sequence encoding a bovine myostatin. The nucleic acid sequence can be that identified as SEQ ID NO:1.
The invention includes a method for identifying a nucleotide sequence of a mutant gene encoding a myostatin protein of a mammal displaying muscular hyperplasia. The method includes obtaining a sample of material containing DNA from the mammal and probing the sample using a nucleic acid probe based on a nucleotide sequence of a known gene encoding myostatin in order to identify a nucleotide sequence of the mutant gene. In a particular approach, the probe is based on a nucleotide sequence identified as SEQ ID NO:1, SEQ ID NO:5 or SEQ ID NO:7. Preferably, the probe is at least 8 nucleotides in length. The step of probing the sample can include exposing the DNA to the probe under hybridizing conditions and further comprising isolated hybridized nucleic acid molecules. The method can further include the step of sequencing isolated DNA. The method can include the step of isolating and sequencing a cDNA or mRNA encoding the complete mutant myostatin protein. The method can include a step of isolating and sequencing a functional wild type myostatin from the mammal not displaying muscular hyperplasia.
The method can include comparing the complete coding sequence of the complete mutant myostatin protein with, if the coding sequence for a functional wild type myostatin from such a mammal is previously known, (1) the known sequence, or if the coding sequence for a functional myostatin from such a mammal is previously unknown, (2) the sequence(s) determined according to the invention, to determine the location of any mutation in the mutant gene.
The invention includes a primer composition useful for the detection of a nucleotide sequence encoding a myostatin containing a first nucleic acid molecule based on a nucleotide sequence located upstream of a mutation determined according to a method of the invention and a second nucleic acid molecule based on a nucleotide sequence located downstream of the mutation.
A probe of the invention can include a nucleic acid molecule based on a nucleotide sequence spanning a mutation determined according to the invention.
The invention includes an antibody to a protein encoded by a nucleotide sequence identified as SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:7, or other protein of the present invention.
The invention includes a transgenic mammal, usually non-human, having a phenotype characterized by muscular hyperplasia, said phenotype being conferred by a transgene contained in the somatic and germ cells of the mammal, the transgene encoding a myostatin protein having a dominant negative mutation. The transgenic animal can be male and the transgene can be located on the Y chromosome. The mammal can be bovine and the transgene can be located to be under the control of a promoter which is normally a promoter of a myosin gene.
Another transgenic mammal, usually non-human, has a phenotype characterized by muscular hyperplasia, in which the phenotype is conferred by a transgene having a sequence antisense to that encoding a myostatin protein of the mammal. The mammal can be a male bovine and the transgene can be located on the Y chromosome. The transgene can further include a sequence which when transcribed obtains an RNA having ribozyme or siRNA (small interfering RNA) activity.
A transgenic non-human mammal of the invention having a phenotype characterized by muscular hyperplasia, can have the phenotype inducible and conferred by a myostatin gene flanked by loxP sites and Cre transgene under the dependence of an inducible promoter.
A transgenic non-human mammal of the invention having a phenotype characterized by muscular hyperplasia, can have the phenotype inducible and conferred by a myostatin gene flanked by loxP sites and Cre transgene located on the Y chromosome.
The invention includes a method for determining whether a sample of mammalian genetic material is capable of conferring a phenotype characterized by muscular hyperplasia, comprising ascertaining whether the genetic material contains a nucleotide sequence encoding a protein having the biological activity of myostatin, wherein the absence of said sequence indicates the presence of muscular hyperplasia in the animal.
An important objective of the instant invention is to provide a transgenic non-human male mammal exhibiting muscular hypertrophy, most particularly, but not limited to, a transgenic bovine.
Another important objective of the instant invention is to provide a method for producing a transgenic non-human male mammal exhibiting muscular hypertrophy, most particularly, but not limited to, a transgenic bovine.
It is also an objective of the invention to provide the embryonic stem cells or somatic cells for nuclear transfer necessary to produce a transgenic non-human male mammal exhibiting muscular hypertrophy. The somatic cells are preferably, but not limited to, fetal fibroblasts.
Other objectives and advantages of this invention will become apparent from the following description taken in conjunction with the accompanying drawings wherein are set forth, by way of illustration and example, certain embodiments of this invention. The drawings constitute a part of this specification and include exemplary embodiments of the present invention and illustrate various objects and features thereof.
This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
In describing particular aspects of the invention, reference is made to the accompanying drawings, in which:
The following list defines terms, phrases and abbreviations used throughout the specification. Although the terms, phrases and abbreviations are listed in the singular tense, this list is intended to encompass all grammatical forms.
As used herein, the term “double-muscling” describes an increase in skeletal muscle mass due to loss of the biological function of myostatin protein. Double-muscling can result from muscular hyperplasia and/or hypertrophy.
As used herein, the term “hyperplasia” refers to an abnormal increase in the number of cells in an organ and/or tissue resulting in enlargement of the organ and/or tissue.
As used herein, the term “hypertrophy” refers to the enlargement of an organ and/or tissue resulting from an increase in the size of the individual cells of the organ and/or tissue.
As used herein, the abbreviation “MSTN” refers to myostatin. Myostatin a protein of the transforming growth factor-(β (TGF-β)superfamily that acts as a negative regulator of skeletal muscle mass (Lee and McPherron PNAS 98:169306-9311 2001). Myostatin is also called growth differentiation factor-8 (GDF-8). Disruption of the myostatin gene in mice double skeletal muscle mass (McPherron et al. Nature 387:83-90 1997). Conversely, systemic over-expression of the myostatin gene leads to a wasting syndrome characterized by extensive muscle loss (Zimmers et al. Science 296:1486-1488 2002).
As used herein, the abbreviation “LAP” refers to the latency-associated peptide or myostatin propeptide. Myostatin protein as purified from mammalian cells consists of a noncovalently held complex of the N-terminal propeptide and a disulfide-linked dimer of C-terminal fragments. This C-terminal dimer is held in an inactive complex with the propeptide and other proteins. Thus, the myostatin propeptide or LAP as an inhibitory/inactivating effect on the biological function of myostatin (Lee and McPherron PNAS 98:169306-9311 2001).
As used herein, the term “dominant-negative effect” involves proteins that act as dimers and results from the ability of a mutated/inactive subunit to dimerize with the active subunit and thus inactivate the normal protein. The transgenic non-human male mammals of the instant invention are produced using the latency-associated peptide as a dominant-negative means to repress endogenous myostatin activity.
As used herein, the phrase “protein having biological activity of myostatin” means that a protein or any portion thereof is capable of the biological function of myostatin.
As used herein, the term “genotype” refers to the entire genetic constitution of an organism; i.e. genes of an organism, both dominant and recessive.
As used herein, the term “phenotype” refers to the observable characteristics of an individual resulting from the interaction of the individual's genotype with the environment. For example, the phenotype of double-muscling is seen in an animal having within its' genotype the nt821(del11) mutation in the gene encoding for myostatin.
As used herein, the term “allele” refers to an alternative form of a gene and/or any one of several mutational forms. The nt821(del11) mutation and the normal are both alleles of the MSTN gene.
As used herein, the term “microsatellite” refers to a segment of DNA about 2 to 6 nucleotides in length which is tandomly repeated.
As used herein, the term “promoter” refers to a sequence at the 5′ end of a gene which binds DNA polymerase and/or transcription factors to regulate expression of the gene. Promoters can be tissue-specific.
As used herein, the term “transgenic” refers to a cell and/or animal having a genome into which genetic material from a different organism has been artificially introduced. The transgenic animals of the instant invention contain DNA for a myostatin trans-repressor that when expressed inactivates endogenous myostatin.
As used herein, the phrase “naturally occurring mutation” refers to a mutation in genetic material that is not artificially introduced.
As used herein, the abbreviation “BBB” refers to the Belgian Blue Breed of cattle. The BBB of cattle express naturally occurring myostatin-inactivating mutations.
As used herein, the term “dystocia” refers to a slow and/or difficult labor. The BBB of cattle often experience dystocia due to the double-muscling phenotype.
As used herein, the term “bovine” means of or relating to an animal of the cattle group, including buffalo and bison.
As used herein, the term “murine” means of or relating to rodents, including mice and rats.
As used herein, the abbreviation “ES cell” refers to an embryonic stem cell which is a pluripotent, balstocyst-derived cell that retains the developmental potential to differentiate into all somatic and germ cell lineages (Robertson, E. J. Trends in Genetics 2:9-13 1986). The ES cells of the instant invention are preferably, but not limited to, murine ES cells.
As used herein, the term “expression” includes transcription and translation.
As used herein, the term “heterologous” or “foreign” refers to nucleic acid and/or amino acid sequences not naturally occurring in the cell/organism of interest. Heterologous sequences may also be found in a location or locations in the genome that differs from that in which it occurs in nature.
As used herein, the term “endogenous” refers to nucleic acid and/or amino acid sequences naturally occurring in the cell/organism of interest.
As used herein, the term “recombinant” refers to genetic material, cells and/or organisms that have been genetically modified; for example, by addition of heterologous genetic material or modification of the endogenous genetic material.
As used herein, the term “isolated” or “purified” refers to nucleic acid and/or amino sequences that have been removed from at least one component with which it is naturally associated. For example, an isolated protein is substantially free of cellular material or culture medium when produced by molecular biological techniques.
As used herein, the term “vector” refers to a polynucleotide construct designed for transduction and/or transfection of one or more cell types.
As used herein, the phrase “operably linked” when referring to a transcriptional regulatory element and a coding sequence is intended to mean that the regulatory sequence is associated with the coding sequence in such a manner as to facilitate transcription of the coding sequence.
As used herein, the term “homologous recombination” refers to the exchange of DNA fragments between two DNA molecules or chromatids at the site of homologous nucleotide sequences.
As used herein, the term “gene targeting” refers to a type of homologous recombination that occurs when a fragment of genomic DNA is introduced into a mammalian cell and that fragment locates and recombines with endogenous homologous sequences. The first step of the method for producing the transgenic non-human male mammals of the invention is carried out by gene targeting.
As used herein, the term “cre recombinase” refers to a specific DNA recombinase which recognizes a specific nucleotide sequence (lox site) and conducts complete processing, including strand cleavage, strand exchange and ligation of each strand within the site. A cre gene can be isolated from the E. coli bacteriophage P1 by methods known in the art (Abremski et al. Cell 32:1301-1311 1983; Sternberg et al. Journal of Molecular Biology 150:467-486 1981). The use of a cre/lox system provides specific gene expression at a specifically desired time.
As used herein, the term “lox site” refers to a specific sequence of nucleotides recognized by a cre recombinase. There are several different lox sites, including, but not limited to, loxP, loxB, loxl, loxR and loxC2. These sequences can be isolated from the E. coli bacteriophage P1 by methods known in the art (Abremski et al. Cell 32:1301-1311 1983; Sternberg et al. Journal of Molecular Biology 150:467-486 1981). The term “flox” means to flank a portion of a nucleotide sequence (gene) with one or more lox sites.
As used herein, the abbreviation “RMCE” refers recombinase-mediated cassette exchange, a method for specific expression of genetic material at a given time mediated by the cre recombinase. The second step of the method for producing the transgenic non-human male mammals of the invention is carried out by RMCE.
As used herein, the term “FISH” refers to fluorescent in situ hybridization, a technique useful for identifying whole chromosomes or parts of chromosomes using fluorescent-tagged DNA probes.
As used herein, the abbreviation “TSPY” refers to the testis-specific protein Y-encoded pseudogene. A pseudogene is a segment of DNA that resembles a gene but lacks a genetic function. The TSPY gene was chosen as the targeting site on the murine Y chromosome for the first step of the claimed method for producing the transgenic male mouse of the instant invention.
As used herein, the abbreviation “MLC” refers to the myosin light chain, specific to skeletal muscle. The regulatory elements (promoter and enhancer) of the MLC gene are used to control the expression of the LAP in the transgenic non-human male mammals of the instant invention.
As used herein, the abbreviation “SV40” refers to the simian virus 40. The regulatory elements (small tumor antigen intron and polyadenylation signal) of the SV40 genome are used to control the expression of the LAP in the transgenic non-human male mammals of the instant invention.
As used herein, the term “polyadenylation signal” refers to a sequence (AATAAA) near the 3′ end of a primary transcript which signals that a polyadenine tail be added to the newly formed transcript. A polyadenine tail can be several hundred nucleotides long and seems to play a role in the stability of mRNA.
As used herein, the abbreviation “BAC” refers to a bacterial artificial chromosome. A BAC is a cloning vector based on E. coli F-factor replicon. Large segments of DNA from another species are cloned into bacterial DNA to form BACs.
As used herein, the term “CpG islands” refers to areas of multiple CG (cytosine and guanine) repeats in a nucleic acid molecule.
As used herein, the term “ESTs” refers to short segments of incompletely sequenced cDNA; allow for design of a PCR reaction which may be used to test for the presence of the cDNA.
As used herein, the abbreviation “UP” means upstream.
As used herein, the abbreviation “BC” means backcross.
As used herein, the abbreviation “CONT” means control.
The method used for isolating genes which cause specific phenotypes is known as positional candidate cloning. It involves: (i) the chromosomal localization of the gene which causes the specific phenotype using genetic markers in a linkage analysis; and (ii) the identification of the gene which causes the specific phenotype amongst the “candidate” genes known to be located in the corresponding region. Most of the time these candidate genes are selected from available mapping information in humans and mice.
The tools required to perform the initial localization (step(i) above) are microsatellite marker maps, which are available for livestock species and are found in the public domain (Bishop et al., 1994; Barendse et al., 1994; Georges et al., 1995; and Kappes, 1997). The tools required for the positional candidate cloning, particularly the YAC libraries, (step (ii) above) are partially available from the public domain. Genomic libraries with large inserts constructed with Yeast Artificial Chromosomes (“YAC”) are available in the public domain for most livestock species including cattle. When cross-referencing the human and mouse map, it is necessary to identify the positional candidate, which is available at low resolution but needs to be refined in every specific instance to obtain the appropriate level of high resolution. A number of original strategies are described herein to achieve this latter objective. For general principles of positional candidate cloning, see Collins, 1995 and Georges and Andersson, 1996.
In order to allow for cross-referencing between the bovine and human gene map as part of the positional candidate cloning approach, HSA2q31-32 (map of the long arm of human chromosome 2, cytogenetic bands q31-32) and BTA2q12-22 (map of the arm of bovine chromosome 2, cytogenetic bands q12-22) were integrated on the basis of coincidence, bovine YAC's as described below.
Using a previously described experimental [(normal x double-muscled) x double-muscled] backcross population comprising 108 backcross individuals, the mh locus was recently mapped by linkage analysis to the centromeric tip of bovine chromosome 2 (BTA2), at 3.1 centiMorgan proximal from the last marker on the linkage map: TGLA44 (Charlier et al., 1995). It was also known from previous work that pro-α(III) collagen (Col3AI) was located in the same chromosomal region as the mh locus. Col3AI has been mapped to BTA2q12-22 by in situ hybridization (Solinas-Toldo et al., 1995), while a Col3AI RFLP marker was shown to be closely linked to TGLA44 (θ=2%)(Fisher et al., 1996). This identifies the region flanking Col3AI on the human map, i.e. HSA2q31-32, as the likely orthologous human chromosome segment. This assumption is compatible with data from Zoo-FISH experiments (Solinas-Toldo et al., 1995) as well as mapping data of Type I markers on somatic cell hybrids (O'Brien et al., 1993), which establish an evolutionary correspondence between segments of HSAq2 and BTA2.
In order to refine the correspondence between the HSA2q31-33 and BTA2q12-22 maps, Comparative Anchored Tagged Sequences or CATS, i.e. primer pairs that would amplify a Sequence Tagged Site or STS from the orthologous gene in different species (Lyons et al., 1996), were developed for a series of genes flanking Col3A1 on the human map and for which sequence information was available in more than one mammal. In addition to Col3AI, working CATS were obtained for α2(V) collagen (Col5A2), inositol polyphosphate-1 phosphatase (INPP1), tissue factor pathway inhibitor precursor (TFPI), titin (TTN), n-chimaerin (CHN), glutamate decarboxylase 67 (GAD1), Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and T-cell membrane glycoprotein CD28 (CD28). The corresponding primer sequences are given in Table 1.
These CATS were used to screen a 6-genome equivalent bovine YAC library by PCR using a three-dimensional pooling strategy as described by Libert et al., 1993. The same YAC library was also screened with all microsatellite markers available for proximal BTA2, i.e. TGLA44, BM81124, BM3627, ILSTS026, INRA40 and TGLA431 (Kappes et al., 1997).
Potential overlap between the YACs obtained with this panel of STS's was explored on the basis of common STS content, as well as cross-hybridization between SINE-PCR product from individual YACs. From this analysis, three independent YAC contigs emerged in the region of interest: (i) contig A containing microsatellites TGLA44, BM81124 and Type I marker INPP1; (ii) contig B containing Col3AI and Col5A2; and (iii) contig C containing microsatellite markers BM3627, ILSTS026 and INRA40, and Type I marker TFPl.
None of the available microsatellites mapped to contig B, therefore this cluster of YACs could not be positioned in cattle with respect to the other two contigs. Available mapping information in the human, however, allowed prediction of contig B's position between contigs A and C. To test this hypothesis, two new microsatellite markers were isolated from contig B, BULGE20 and BULGE28. BULGE20 proved to be polymorphic, allowing for genotyping of the experimental backcross population.
In addition, to increase the informativeness of the markers available for contig A, two new microsatellite markers were developed from this contig: BULGE23 and BULGE27. BULGE23 proved to be polymorphic and was used to type the same pedigree material.
All resulting genotypes were used to construct a linkage map using the lLINK program (Lathrop and Lalouel, 1984). The following most likely order and sex-averaged recombination rates between adjacent markers was obtained: [TGLA44-(0%)-BULG23]-(6.1%)-BULG20-(1.6%)-ILSTS026-(2.3%)-INRA40-(7.1%)-TGLA431. The position of BULGE20 between TGLA44 and ILSTS026 confirmed the anticipated order of the three contigs.
A multi point linkage analysis was undertaken using LINKMAP, to position the mh locus with respect to the new marker map. Linkage analysis was performed under a simple recessive model, assuming full penetrance for mh/mh individuals and zero penetrance for the two other genotypes. The LOD score curve shown in
Recently, McPherron et al. (1997) demonstrated that mice homozygous for a knock-out deletion of GDF-8 or myostatin, were characterized by a generalized increase in skeletal muscle mass. Using the published 2676 bp murine myostatin cDNA sequence (GenBank accession number U84005), a Tentative Human Consensus (THC) cluster in the Unigene database was identified which represented three cDNA clones (221299, 300367, 308202) and six EST (Expressed Sequence Tag) sequences (H92027, H92028, N80248, N95327, W07375, W24782). The corresponding THC covered 1296 by of the human myostatin gene, showing an homology of 78.1% with the murine sequence when averaged over the entire sequence, and 91.1% when considering only the translated parts of the human and murine genes (566 bp). This THC therefore very likely corresponds to the human orthologue of the murine myostatin gene.
Primers (5′-GGCCCAACTATGGATATATTTG-3′ (SEQ ID NO:9) and 5′-GGTCCTGGGAAGGTTACAGCA-3′ (SEQ ID NO:10)) were thus prepared to amplify a 272 by fragment from the second exon of human myostatin and used to genotype the whole-genome Genebridge-4 radiation hybrid panel (Walter et al., 1994). The RHMapper program (Slonim et al., unpublished) was used to position the myostatin gene with respect to the Whitehead/MIT framework radiation hybrid map, placing it at position 948.7 cR of the HSA2 map with an associated lodscore >3. Closer examination of the myostatin segregation vector and its confrontation with the vectors from all markers located in that region (Data Release 11.9, May 1997) showed it to be identical to EST SGC38239 placed on the Whitehead/MIT radiation hybrid map (Hudson et al., 1995) at position 946.8 cR of HSA2. This places the human myostatin gene on the RH-map in the interval between Col3AI (EST WI16343-942.5 cR) and INPP1 (EST L08488-950.2 to 951.2 cR) (
To test the potential involvement of myostatin in the determinism of muscling in cattle, primer pairs were designed based on the available mouse and human myostatin sequence, with the objective to amplify the entire coding sequence from bovine cDNA using PCR (Polymerase Chain Reaction). Whenever possible, primers were therefore positioned in portions of the myostatin sequence showing 100% homology between mouse and human. Two primer pairs were identified that amplified what was predicted to represent 98.4% DNA fragments, respectively 660 (primers GDF8.19-GDF8.12) and 724 by (primers GDF8.11-GDF8.21) long. The expected DNA products were successfully amplified from cDNA generated from skeletal muscle of both a normal (homozygous +/+) (SEQ ID NO:1) and a double-muscled (homozygous mh/mh) (SEQ ID NO:3) animal, and cycle-sequences on both strands.
The nucleotide sequence corresponding to the normal allele presented 88.9% identity with the mouse myostatin sequence (SEQ ID NO:5) over a 1067 by overlap, and contained the expected open reading frame encoding a protein (SEQ ID NO:2) showing 92.9% identity in a 354 amino-acid overlap with mouse myostatin (SEQ ID NO:6). As expected for a member of the TGF superfamily, the bovine myostatin gene is characterized by a proteolytic processing site thought to mediate cleavage of the bioactive carboxy-terminal domain from the longer N-terminal fragment, and by nine cysteine residues separated by a characteristic spacing and suspected to be involved in intra- and inter-molecular disulfide bridges (McPherron and Lee, 1996).
The nucleotide sequence obtained from the mh allele was identical to the normal allele over its entire length, except for an 11 bp deletion involving nucleotides 821 to 831 (counting from the initiation codon). This frame shifting deletion, occurring after the first cysteine residue of the carboxy-terminal domain, drastically disrupts the downstream amino-acid sequence and reveals a premature stop-codon after 13 amino acids, see
To further strengthen the assumption of the causality of this mutation, primer pairs flanking the deletion (
To examine the distribution of the nt821(del11) mutation in different conventional and double-muscled breeds, a cohort of 25 normal individuals were genotyped representing two dairy breeds (Holstein-Friesian, Red-and -White) and a cohort of 52 double-muscled animals representing four breeds (BBCB, Asturiana, Maine-Anjou and Piémontese). The results are summarized in Table 2. All dairy animals were homozygous normal except for one Red-and-White bull shown to be heterozygous. The occurrence of a small fraction of individuals carrying the mutation in dairy cattle is not unexpected as the phenotype is occasionally described in this breed. In BBCB and Asturiana, all double-muscled animals were homozygous for the nt821(del11) deletion, pointing towards allelic homogeneity in these two breeds. Double-muscled Maine-Anjou and Piémontese animals were homozygous “normal”, i.e. they did not show the nt821(del11) deletion but a distinct cysteine to tyrosine substitution (C313Y) in double-muscled Piédmontese animals identified by others (Kambadur et al., 1997) was discovered.
The entire coding sequence was also determined for the myostatin gene in double-muscled individuals from ten European cattle breeds and a series of mutation that disrupt myostatin function were identified.
The coding sequence of four control Holstein-Friesian and Jersey individuals was identical to the previously described wild-type allele (Grobet et al., 1997), further indicating that it was the genuine myostatin coding sequence being amplified, and not a non-functional pseudogene.
Amongst the 32 double-muscled animals, seven DNA sequence variants within the coding region were found, as summarized in
In addition to the nt821(del11) mutation in the third exon, described above, four new mutations that would be expected to disrupt the myostatin function were found. An insertion/deletion at position 419 counting from the initiation codon, replacing 7 base pairs with an apparently unrelated stretch of 10 base pairs, reveals a premature stop codon in the N-terminal latency-associated peptide at amino-acid position 140. This mutation is referred to as nt419(de17-ins10). Two base pair substitutions in the second exon, a C→T transition at nucleotide position 610 and a G→T transversion at nucleotide position 676, each yield a premature stop codon in the same N-terminal latency-associated peptide at amino-acid positions 204 and 226 respectively. These mutations are called Q204X and E226X respectively. Finally, a G→A transition at nucleotide position 938 results in the substitution of a cysteine by a tyrosine. This mutation is referred to as C313Y. This cysteine is the fifth of nine highly conserved cysteine residues typical of the members of the TGF-β superfamily and shared in particular by TGF-β1, -β2 and -β3, and inhibin-βA and -βB (McPherron & Lee, 1996). It is thought to be involved in an intramolecular disulfide bridge stabilizing the three-dimensional conformation of the bioactive carboxyterminal peptide. Its substitution is therefore likely to affect the structure and function of the protein. This C313Y has recently also been described by Kambadur et al. (1997).
A conservative phenylalanine to leucine substitution was also found at amino acid position 94 in the first exon, due to a C→A transversion at nucleotide position 282 of the myostatin gene. Given the conservative nature of the amino acid substitution, its location in the less conserved N-terminal latency-associated peptide, and as this mutation was observed at the homozygous condition in animals that were not showing any sign of exceptional muscular development, this mutation probably does not interfere drastically with the myostatic function of the encoded protein, if at all. This mutation is referred to as F94L. The murine protein is characterized by a tyrosine at the corresponding amino acid position.
Also identified was a silent C→T transition at the third position of the 138th cytosine codon in the second exon, referred to as nt414(C-T).
In addition to these DNA sequence polymorphisms detected in the coding region of the myostatin gene, also found were four DNA sequence variants in intronic sequences which are probably neutral polymorphisms and which have been assigned the following symbols: nt374-51(T-C), nt374-50(G-A), nt374-16(del1) in intron 1, and nt748-78(del1) in intron 2 (
Only in Limousin and Blonde d'Aquitaine was there no clear evidence for the role of myostatin loss-of-function mutations in the determinism of the observed muscular hypertrophy. Most Limousin animals were homozygous for the conservative F94L substitution which is unlikely to cause the muscular hypertrophy characterizing these animals, as discussed above. One Limousin animal proved to be heterozygous for this mutation, the other allele being the “wild-type” one. All Blonde d'Aquitaine animals were homozygous “wild-type”. These data indicate either that the myostatin gene is possibly not involved in the double-muscled condition characterizing these two breeds, or that there are additional myostatin mutations outside of the coding region. The double-muscling condition is often considered to be less pronounced in Limousin animals compared to other breeds.
The data indicate that some mutations, such as the nt821del(11) and C313Y, are shared by several breeds which points towards gene migration between the corresponding populations, while others seems to be confined to specific breeds. Moreover, while some breeds (the Belgian Blue breed in particular) seem to be essentially genetically homogeneous others show clear evidence for allelic heterogeneity (e.g. Maine-Anjou).
The observation of allelic heterogeneity contradicts with the classical view that a single mh mutation spread through the European continent in the beginning of the 19th century with the dissemination of the Shorthorn breed from the British Isles (Ménissier, 1982). Two of the mutations at least are shared by more than one breed, indicating some degree of gene migration but definitely not from a single origin.
In mice, and in addition to the in vitro generated myostatin knock-out mice (McPherron and Lee, 1997), the compact mutation can be due to a naturally occurring mutation at the myostatin gene. The compact locus has been mapped to the D1Mit375-D1Mit21 interval on mouse chromosome 1 known to be orthologous to HSA2q31-32 and BTA2q12-22 (Varga et al., 1997).
From an applied point of view, the characterization of a panel of mutations in the myostatin gene associated with double-muscling contributes to the establishment of a diagnostic screening system allowing for marker-assisted selection for or against this condition in cattle.
Integration of the HSA2q31-32 and BTA2q12-22 maps were done by using coincident YAC's and the mh locus was positioned in the interval flanked by Col3AI and INPP1 as follows. Genetic mapping was performed using a previously described (Holstein-Friesian×Belgian Blue)×Belgian Blue experimental backcross population counting 108 informative individuals (Charlier et al., 1995). Microsatellite genotyping was performed according to standard procedures (Georges et al., 1995), using the primer sequences reported in Table 1. Linkage analyses were performed with the MLINK, ILINK and LINKMAP programs of the LINKAGE (version 5.1) and FASTLINK (2.3P version, June 1995) packages (Lathrop & Lalouel, 1984; Cottingham et al., 1993). The YAC library was screened by PCR using a three dimensional pooling scheme as described in Libert et al., 1993. The primer pairs corresponding to the CATS used to screen the library are reported in Table 1. Cross-hybridization between SINE-PCR products of individual YACs was performed according to Hunter et al. (1996), using primers reported in Lenstra et al. (1993). Microsatellites were isolated from Yacs according to Cornelis et al. (1992).
DNA from the Genebridge-4-panel (Walter et al., 1994) was purchased from Research Genetics (Huntsville, Ala.), and genotyped by PCR using standard procedures and the following human myostatin primer pair (5′GGCCCAACTATGGATATATTTG-3′ and 5′-GGTCCTGGGAAGGTTACAGCA-3′, SEQ ID NOS: 9 and 10 respectively). Mapping was performed via the WWW server of the Whitehead Institute/MIT Center for Genome Research using their RH-mapper program (Slonim, D.; Stein, L.; Kruglyak, L.; Lander, E., unpublished) to position the markers with respect to the framework map. Segregation vectors of the query markers were compared with the vectors from all markers in the region of interest in the complete Data Release 11.9 (May 1997) to obtain a more precise position. This positions myostatin in the INPP1-Colb 3AI on the human map with LOD score superior to 3.
To clone the bovine myostatin orthologue a strategy based on RT-PCR amplification from skeletal muscle cDNA was chosen. Total RNA was extracted from skeletal muscle (Triceps brachialis) according to Chirgwin et al. (1979). RT-PCR was performed using the Gene-Amp RNA PCR Kit (Perkin-Elmer) and the primers reported in Table 1. The PCR products were purified using QiaQuick PCR Purification kit (Qiagen) and sequenced using Dye terminator Cycle Sequencing Ready Reaction (Perkin-Elmer) and an ABI373 automatic sequencer, using the primers reported in Table 2.
Diagnosis of the nt821(del11) Deletion
To diagnose the nt821(del11) the following primer sequences were designed flanking the nt821(del11) deletion: 5′-TCTAGGAGAGATTTTGGGCTT-3′ (SEQ ID NO:68) and 5-GATGGGTATGAGGATACTTTTGC-3′ (SEQ ID NO:69). These primers amplify a 188 by fragments from normal individuals and a 177bp fragment from double-muscled individuals. Heterozygous individuals show the two amplification products. These amplification products can be detected using a variety of methods. In this example the PCR product was labeled by incorporation of dCTP32, separated on a denaturing acrylamide gel and revealed by autoradiography. Other approaches that could be used to distinguish the three different genotypes are known to those skilled in the art and would include separation in agarose gels and visualization with ethidium bromide, direct sequencing, TaqMan assays, hybridization with allele specific oligonucleotides, reverse dot-blot, RFLP analysis and several others. The specificity of the test is linked to the detected mutation and not to the primers used in the detection method. That means that other primers can easily be designed based on said bovine myostatin sequence that would fulfill the same requirements.
A total of 32 animals with extreme muscular development were sampled from ten European beef cattle breeds in which double-muscled animals are known to occur at high to moderate frequency: (i) Belgium: Belgian Blue (4), (ii) France: Blonde d'Aquitaine (5), Charolais (2), Gasconne (2), Limousin (5), Maine-Anjou (4), Parthenaise (3), (iii) Spain: Asturiana (2), Rubia Gallega (2), (iv) Italy: Piedmontese (2). The determination of the double-muscled phenotype of the sampled animals was performed visually by experienced observers. Four animals with conventional phenotype sampled from the Holstein-Friesian (2) and Jersey (2) dairy populations were analyzed as controls.
In order to facilitate the study of the myostatin coding sequence from genomic DNA, the sequences of the exon-intron boundaries of the bovine gene were determined. In mice, the myostatin gene is known to be interrupted by two introns, respectively ≈1.5 and 2.4 Kb long (McPherron & Lee, 1997). Two primer pairs were thus designed, respectively, in bovine exons 1 and 2, and exons 2 and 3, that were predicted to flank the two introns, assuming conservation of gene organization between mouse and cattle (
Reading from left to right and down the table the sequences given in Table 3 are identified as follows: Intron 1-5′ is SEQ ID NO:1(positions 365-391), SEQ ID NO:3(positions 365-391) and SEQ ID NO:54 (positions 772-798); Intron 1-3′ is SEQ ID NO:70; Intron 2-5′ is SEQ ID NO:7l; Intron 2-3′ is SEQ ID NO:72; Exon 1-5′ is SEQ ID NO:54 (positions 324-354); Exon 1-3′ is SEQ ID NO: 73, Exon 2-5′ is SEQ ID NO:54 (positions 2574-2600); Exon 2-3′ is SEQ ID NO:74; Exon 3-5′ is SEQ ID NO:54 (positions 4952-4978); Exon 3-3′is SEQ ID NO:75; Exon 1-seq1 is SEQ ID NO:76; Exon 1-seq2 is SEQ ID NO:1 (positions 209-231), SEQ ID NO:3 (positions 209-231) and SEQ ID NO:54 (positions 616-638); Exon 2-seq1 is SEQ ID NO:77; Exon 2-seq 2 is SEQ ID NO:78; Exon 2-seq3 is SEQ ID NO:1 (positions 594-620), SEQ ID NO:3 (positions 594-620) and SEQ ID NO:54 (positions 2827-2853); Exon 3-seq1 is SEQ ID NO:79; Exon 3-seq2 is SEQ ID NO:1 (positions 1039-1063), SEQ ID NO:3 (positions 1028-1052) and SEQ ID NO:54 (positions 5304-5328).
Based on the available exonic and intronic sequences of the bovine myostatin gene, three primer pairs that jointly allow for convenient amplification of the entire coding sequence from genomic DNA were designed. The position of the corresponding primers is shown in
After PCR amplification of the entire coding sequence from genomic DNA in the three described fragments, these were purified using QiaQuick PCR purification kit (Qiagen) and sequenced using Dye terminator Cycle Sequencing Ready Reaction (Perkin-Elmer) and an AB1373 automatic sequencer, using the primers used for amplification as well as a series of nested primers (
Nickerson, personal communication), which is part of a series of sequence analysis programs including Phred (Ewing, B. & Green, P. (1992), unpublished), Phrap (Green, P. (1994), unpublished) and Consed (Gordon, D. (1995), unpublished), but any suitable sequencing program would do, as known to a person skilled in the art.
Monoclonal antibodies (Mab's) specific for myostatin are useful. In the case of the bovine protein having the amino acid sequence identified as SEQ ID NO:2, for example, antibodies can be used for diagnostic purposes such as for determining myostatin protein levels in muscle tissue. To produce these antibodies, purified myostatin is prepared. The myostatin can be produced in bacterial cells as a fusion protein with glutathione-S-transferase using the vector pGEX2 (Pharmacia). This permits purification of the fusion protein by GSH affinity chromatography. In another approach, myostatin is expressed as a fusion protein with the bacterial maltose binding domain. The fusion protein is thus recovered from bacterial extracts by passing the extract over an amylose resin column followed by elution of the fusion protein with maltose. For this fusion construct, the vector pMalC2, commercially available from New England Biolabs, can be used. The preparation of a second fusion protein is also useful in the preliminary screening of MAb's.
The generation of hybridomas expressing monoclonal antibodies recognizing myostatin protein is carried out as follows: BALB/c mice are injected intraperitoneally with protein/adjuvant three times at one-month intervals, followed by a final injection into the tail vein shortly prior to cell fusion. Spleen cells are harvested and fused with NS-1 myeloma cells (American Type Culture Collection, Manassas, Va.) using polyethylene glycol 4000 according to standard protocols (Kennett, 1979; Mirski, 1989). The cell fusion process is carried out as described in more detail below.
The fused cells are plated into 96-well plates with peritoneal exudate cells an irradiated spleen cells from BALB/Cc mice as feeder layers and selection with hypoxanthine, aminopterin, and thymidine (HAT medium) is performed.
An ELISA assay is used as an initial screening procedure. 1-10 μg of purified myostatin (cleaved from the fusion protein) in PBS is used to coat individual wells, and 50-100 μl per well of hybridoma supernatants is incubated. Horseradish peroxidase-conjugated anti-mouse antibodies are used for the colorimetric assay.
Positive hybridomas are cloned by limiting-dilution and grown to large-scale for freezing and antibody production. Various positive hybridomas are selected for usefulness in western blotting and immunohistochemistry, as well as for cross reactivity with myostatin proteins from different species, for example the mouse and human proteins.
Alternatively, active immunization by the generation of an endogenous antibody by direct exposure of the host animal to small amounts of antigen can be carried out. Active immunization involves the injection of minute quantities of antigen (g) which probably will not induce a physiological response and will be degraded rapidly. Antigen will only need to be administered as prime and boost immunizations in much the same manner as techniques used to confer disease resistance (Pell et al., 1997).
Antisense nucleic acids or oligonucleotides (RNA or preferably DNA) can be used to inhibit myostatin production in order to increase muscle mass of an animal. Antisense oligonucleotides, typically 15 to 20 bases long, bind to the sense mRNA or pre mRNA region coding for the protein of interest, which can inhibit translation of the bound mRNA to protein. The cDNA sequence encoding myostatin can thus be used to design a series of oligonucleotides which together span a large portion, or even the entire cDNA sequence. These oligonucleotides can be tested to determine which provides the greatest inhibitory effect on the expression of the protein (Stewart, 1996). The most suitable mRNA target sites include 5′- and 3′-untranslated regions as well as the initiation codon. Other regions might be found to be more or less effective. Alternatively, an antisense nucleic acid or oligonucleotide may bind to myostatin coding or regulatory sequences.
Rather than reducing myostatin activity by inhibiting myostatin gene expression at the nucleic acid level, activity of the myostatin protein may be directly inhibited by binding to an agent, such as, for example, a suitable small molecule or a monoclonal antibody.
It will of course be understood, without the intention of being limited thereby, that a variety of substitutions of amino acids is possible while preserving the structure responsible for myostatin activity of the proteins disclosed herein. Conservative substitutions are described in the patent literature, as for example, in U.S. Pat. Nos. 5,264,558 or 5,487,983. It is thus expected, for example, that interchange among non-polar aliphatic neutral amino acids, glycine, alanine, proline, valine and isoleucine, would be possible. Likewise, substitutions among the polar aliphatic neutral amino acids, serine, threonine, methionine, asparagine and glutamine could possibly be made. Substitutions among the charged acidic amino acids, aspartic acid and glutamic acid, could probably be made, as could substitutions among the charged basic amino acids, lysine and arginine. Substitutions among the aromatic amino acids, including phenylalanine, histidine, tryptophan and tyrosine would also likely be possible. These sorts of substitutions and interchanges are well known to those skilled in the art. Other substitutions might well be possible. Of course, it would also be expected that the greater the percentage of homology, i.e., sequence similarity, of a variant protein with a naturally occurring protein, the greater the retention of metabolic activity. Of course, as protein variants having the activity of myostatin as described herein are intended to be within the scope of this invention, so are nucleic acids encoding such variants.
A further advantage may be obtained through chimeric forms of the protein, as known in the art. A DNA sequence encoding the entire protein, or a portion of the protein, could thus be linked, for example, with a sequence coding for the C-terminal portion of E. coli β-galactosidase to produce a fusion protein. An expression system for human respiratory syncytial virus glycoproteins F and G is described in U.S. Pat. No. 5,288,630 issued Feb. 22, 1994 and references cited therein, for example.
A recombinant expression vector of the invention can be a plasmid, as described above. The recombinant expression vector of the invention further can be a virus, or portion thereof, which allows for expression of a nucleic acid introduced into the viral nucleic acid. For example, replication defective retroviruses, adenoviruses and adeno-associated viruses can be used.
The recombinant expression vectors of the invention can be used to make a transformant host cell including the recombinant expression vector. The term “transformant host cell” is intended to include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention. The terms “transformed with”, “transfected with”, “transformation” and “transfection” are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art. Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium-chloride mediated transformation. Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, electroporation or microinjection. Suitable methods for transforming and transfecting host cells are known (Sambrook, 1989).
The number of host cells transformed with a recombinant expression vector of the invention by techniques such as those described above will depend upon the type of recombinant expression vector used and the type of transformation technique used. Plasmid vectors introduced into mammalian cells are integrated into host cell DNA at only a low frequency. In order to identify these integrants, a gene that contains a selectable marker (e.g. resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to certain drugs, such as G418 and hygromycin. Selectable markers can be introduced on a separate plasmid from the nucleic acid of interest or, preferably, are introduced on the same plasmid. Host cells transformed with one or more recombinant expression vectors containing a nucleic acid of the invention and a gene for a selectable marker can be identified by selecting for cells using the selectable marker. For example, if the selectable marker encodes a gene conferring neomycin resistance (such as pRc/CMV), transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die.
Nucleic acids which encode myostatin proteins can be used to generate transgenic animals. A transgenic animal (e.g., a mouse) is an animal having cells that contain a transgene, which transgene is introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, a bovine cDNA, comprising the nucleotide sequence shown in SEQ ID NO:1, or an appropriate variant or subsequence thereof, can be used to generate transgenic animals that contain cells which express bovine myostatin. Likewise, variants such as mutant genes (e.g. SEQ ID NO:3) can be used to generate transgenic animals. This could equally well be done with the human myostatin protein and variants thereof. “Knock out” animals, as described above, can also be generated. Methods for generating transgenic animals, particularly animals such as mice, have become conventional in the art are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009. In a preferred embodiment, plasmids containing recombinant molecules of the invention are microinjected into mouse embryos. In particular, the plasmids are microinjected into the male pronuclei of fertilized one-cell mouse eggs; the injected eggs are transferred to pseudo-pregnant foster females; and, the eggs in the foster females are allowed to develop to term. (Hogan,1986). Alternatively, an embryonal stem cell line can be transfected with an expression vector comprising nucleic acid encoding a myostatin protein, and cells containing the nucleic acid can be used to form aggregation chimeras with embryos from a suitable recipient mouse strain. The chimeric embryos can then be implanted into a suitable pseudopregnant female mouse of the appropriate strain and the embryo brought to term. Progeny harboring the transfected DNA in their germ cells can be used to breed uniformly transgenic mice.
Such animals can be used to determine whether a sequence related to an intact myostatin gene retains biological activity of myostatin. Thus, for example, mice in which the murine myostatin gene has been knocked out and containing the nucleic acid sequence identified as SEQ ID NO:1 could be generated along with animals containing the nucleic acid sequence identified as SEQ ID NO:3. The animals could be examined for display of muscular hyperplasia, especially in comparison with knockout mice, which are known to display such. In this way it can be shown that the protein encoded by SEQ ID NO:3 lacks myostatin activity within the context of this invention while the protein encoded by the nucleic acid sequence identified as SEQ ID NO:1 possesses biological activity of myostatin.
In such experiments, muscle cells would be particularly targeted for myostatin (and variants) transgene incorporation by use of tissue specific enhancers operatively linked to the encoding gene. For example, promoters and/or enhancers which direct expression of a gene to which they are operatively linked preferentially in muscle cells can be used to create a transgenic animal which expresses a myostatin protein preferentially in muscle tissue. Transgenic animals that include a copy of a myostatin transgene introduced into the germ line of the animal at an embryonic stage can also be used to examine the effect of increased myostatin expression in various tissues.
The pattern and extent of expression of a recombinant molecule of the invention in a transgenic mouse is facilitated by fusing a reporter gene to the recombinant molecule such that both genes are co-transcribed to form a polycistronic mRNA. The reporter gene can be introduced into the recombinant molecule using conventional methods such as those described in Sambrook et al., (Sambrook, 1989). Efficient expression of both cistrons of the polycistronic mRNA encoding the protein of the invention and the reporter protein can be achieved by inclusion of a known internal translational initiation sequence such as that present in poliovirus mRNA. The reporter gene should be under the control of the regulatory sequence of the recombinant molecule of the invention and the pattern and extent of expression of the gene encoding a protein of the invention can accordingly be determined by assaying for the phenotype of the reporter gene. Preferably the reporter gene codes for a phenotype not displayed by the host cell and the phenotype can be assayed quantitatively. Examples of suitable reporter genes include lacZ (β-galactosidase), neo (neomycin phosphotransferase), CAT (chloramphenicol acetyltransferase) dhfr (dihydrofolate reductase), aphIV (hygromycin phosphotransferase), lux (luciferase), uidA (β-glucuronidase). Preferably, the reporter gene is lacZ which codes for β-galactosidase. β-galactosidase can be assayed using the lactose analogue X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) which is broken down by β-galactosidase to a product that is blue in color (Old).
The present invention includes knocking out wild type myostatin in mammals, in order to obtain the desired effect(s) thereof. This is particularly true in cattle raised for beef production. It may well prove advantageous to substitute a defective gene (e.g. SEQ ID NO:3 or its genomic analogue) rather than delete the entire sequence of DNA encoding for a protein having myostatin activity. A method of producing a transgenic bovine or transgenic bovine embryo is described in U.S. Pat. No. 5,633,076, issued May 27, 1997, for example.
The transgenic animals of the invention can be used to investigate the molecular basis of myostatin action. For example, it is expected that myostatin mutants in which one or more of the conserved cysteine residues has been deleted would have diminished activity in relation to a wild type myostatin protein in which all such residues are retained. Further, deletion of proteolytic cleavage site would likely result in a mutant lacking biological activity of myostatin.
Transgenesis can be used to inactivate myostatin activity. This could be achieved by using conventional transgenesis, i.e. by injection in feritilized oocytes, or by gene targeting methods using totipotent cell lines such as ES (embryonic stem cells) which can then be injected in oocytes and participate in the development of the resulting organisms or whose nucleus can be transferred into unfertilized oocytes, nucleus transfer or cloning.
It is also possible to create a genetically altered animal in which the double-muscling trait is dominant so that the animal would be more useful in cross-breeding. Further, in a particular aspect, the dominant trait would be male specific. In this way, bulls would be double-muscled but cows would not be. In addition, or alternatively, the trait would also be unexpressed until after birth or inducible. If inducible the trait could be induced after birth to avoid the calving difficulties described above.
There are at least three approaches that can be taken to create a dominant “mh” allele. Because functional myostatin, a member of the TGF-8 superfamily, is a dimer, dominant negative myostatin mutations can be created (Herskowitz et al., 1987; Lopez et al., 1992). According to one method, this is accomplished by mutating the proteolytic processing site of myostatin. To enhance the dominant negative effect, the gene can be put under the control of a stronger promoter such as the CMV promoter or that of a myosin gene, which is tissue specific, i.e., expressed only in skeletal muscle. Alternatively, an antisense sequence of that encoding myostatin could be incorporated into the DNA, so that complementary mRNA molecules are generated, as understood by a person skilled in the art. Optionally, a ribozyme could be added to enhance mRNA breakdown. In another approach, cre recombinase generate/ribozyme approach or the Cre-lox P system could be used (Hoess et al., 1982; Gu et al., 1994).
Male Specificity Can be Achieved by Placing the Dominant mh Alleles on the Y Chromosome by Homologous Recombination.
Inducibility can be achieved by choosing promoters with post-natal expression in skeletal muscle or using inducible systems such the Tet-On and Tet-Off (Gossen et al. 1992; Shockett et al. 1996).
Using conventional transgenesis a gene coding for a myostatin antisense is injected, for example, by inverting the orientation of the myostain gene in front of its natural promoter and enhancer sequences. This is followed by injection of a gene coding for an anti-myostain ribozyme, i.e. an RNA that would specifically bind to endogenous myostain mRNA and destroy it via its “ribozyme” activity.
Also, through gene targeting, a conventional knock-out animal can be generated, specific mutations by gene replacement can be engineered. It is possible to inactivate the myostain gene at a specific developmental time, such as after birth to avoid calving difficulties. As mentioned above, this could be achieved using the Cre-lox P systems in which l.ox P sides are engineered around the myostain gene by homologous recombination (gene targeting), and mating these animals with transgenic animals having a Cre transgene (coding for the Cre recombinase existing DNA flanked by loxP sides) under the dependence of a skeletal muscle specific promoter only active after birth. This is done to obtain individuals that would inactivate their myostain gene after birth. As mentioned above, there are also gene targeting systems that allow genes to be turned on and off by feeding an animal with, for example, an antibiotic. In such an instance, one engineers an operator between the promoter of the gene and the gene itself. This operator is the target of a repressor which when binding inactivates the gene (for example, the lac operon in E. coli). The repressor is brought into the cell using conventional transgenesis, for example, by injection of the gene coding for the repressor.
Transgenic animals of the invention can also be used to test substances for the ability to prevent, slow or enhance myostatin action. A transgenic animal can be treated with the substance in parallel with an untreated control trangenic animal.
The antisense nucleic acids and oligonucleotides of the invention are useful for inhibiting expression of nucleic acids (e.g. mRNAs) encoding proteins having myostatin activity.
The isolated nucleic acids and antisense nucleic acids of the invention can be used to construct recombinant expression vectors as described previously. These recombinant expression vectors are then useful for making transformant host cells containing the recombinant expression vectors, for expressing protein encoded by the nucleic acids of the invention, and for isolating proteins of the invention as described previously. The isolated nucleic acids and antisense nucleic acids of the invention can also be used to construct transgenic and knockout animals as described previously.
The isolated proteins of the invention are useful for making antibodies reactive against proteins having myostatin activity, as described previously. Alternatively, the antibodies of the invention can be used to isolate a protein of the invention by standard immunoaffinity techniques. Furthermore, the antibodies of the invention, including bi-specific antibodies are useful for diagnostic purposes.
Molecules which bind to a protein comprising an amino acid sequence shown in SEQ ID NO:2 can also be used in a method for killing a cell which expresses the protein, wherein the cell takes up the molecule, if for some reason this were desirable. Destruction of such cells can be accomplished by labeling the molecule with a substance having toxic or therapeutic activity. The term “substance having toxic or therapeutic activity” as used herein is intended to include molecules whose action can destroy a cell, such as a radioactive isotope, a toxin (e.g. diphtheria toxin or ricin), or a chemotherapeutic drug, as well as cells whose action can destroy a cell, such as a cytotoxic cell. The molecule binding to the myostatin can be directly coupled to a substance having a toxic or therapeutic activity or may be indirectly linked to the substance. In one example, the toxicity of the molecule taken up by the cell is activated by myostatin protein.
The invention also provides a diagnostic kit for identifying cells comprising a molecule which binds to a protein comprising an amino acid sequence shown in SEQ ID NO:2, for example, for incubation with a sample of tumor cells; means for detecting the molecule bound to the protein, unreacted protein or unbound molecule; means for determining the amount of protein in the sample; and means for comparing the amount of protein in the sample with a standard. Preferably, the molecule is a monoclonal antibody. In some embodiments of the invention, the detectability of the molecule which binds to myostatin is activated by said binding (e.g., change in fluorescence spectrum, loss of radioisotopic label). The diagnostic kit can also contain an instruction manual for use of the kit.
The invention further provides a diagnostic kit for identifying cells comprising a nucleotide probe complementary to the sequence, or an oligonucleotide fragment thereof, shown in SEQ ID NO:1, for example, for hybridization with mRNA from a sample of cells, e.g., muscle cells; means for detecting the nucleotide probe bound to mRNA in the sample with a standard. In a particular aspect, the invention is a probe having a nucleic acid molecule sufficiently complementary with a sequence identified as SEQ ID NO:1, or its complement, so as to bind thereto under stringent conditions. “Stringent hybridization conditions” takes on its common meaning to a person skilled in the art here. Appropriate stringency conditions which promote nucleic acid hybridization, for example, 6× sodium chloride/sodium citrate (SSC) at about 45° C. are known to those skilled in the art. The following examples are found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6: For 50 ml of a first suitable hybridization solution, mix together 24 ml formamide, 12 ml 20×SSC, 0.5 ml 2 M Tris-HCl pH 7.6, 0.5 ml 100× Denhardt's solution, 2.5 ml deionized H2O, 10 ml 50% dextran sulfate, and 0.5 ml 10% SDS. A second suitable hybridization solution can be 1% crystalline BSA (fraction V), 1 mM EDTA, 0.5 M Na2HPO4 pH 7.2, 7% SDS. The salt concentration in the wash step can be selected from a low stringency of about 2×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. Both of these wash solutions may contain 0.1% SDS. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions, at about 65° C. The cited reference gives more detail, but appropriate wash stringency depends on degree of homology and length of probe. If homology is 100%, a high temperature (65° C. to 75° C.) may be used. If homology is low, lower wash temperatures must be used. However, if the probe is very short (<100bp), lower temperatures must be used even with 100% homology. In general, one starts washing at low temperatures (37° C. to 40° C.), and raises the temperature by 3-5° C. intervals until background is low enough not to be a major factor in autoradiography. The diagnostic kit can also contain an instruction manual for use of the kit.
The invention also provides a diagnostic kit which can be used to determine the genotype of mammalian genetic material, for example. One kit includes a set of primers used for amplifying the genetic material. A kit can contain a primer including a nucleotide sequence for amplifying a region of the genetic material containing one of the naturally occurring mutations described herein. Such a kit could also include a primer for amplifying the corresponding region of the normal gene that produces functional myostatin. Usually, such a kit would also include another primer upstream or downstream of the region of interest complementary to a coding and/or non-coding portion of the gene. A particular kit includes a primer selected from a non-coding sequence of a myostatin gene. Examples of such primers are provided in Table 3, designated as Exon1-5′, Exon1-3′, Exon2-5′, Exon3-5′ and Exon3-3′. These primers are used to amplify the segment containing the mutation of interest. The actual genotyping is carried out using primers that target specific mutations described herein and that could function as allele-specific oligonucleotides in conventional hybridization, Taqman assays, OLE assays, etc. Alternatively, primers can be designed to permit genotyping by microsequencing.
One kit of primers thus includes first, second and third primers, (a), (b) and (c), respectively. Primer (a) is based on a region containing a myostatin mutation, for example a region of the myostatin gene spanning the nt821del(11) deletion. Primer (b) encodes a region upstream or downstream of the region to be amplified by primer (a) so that genetic material containing the mutation is amplified, by PCR, for example, in the presence of the two primers. Primer (c) is based on the region corresponding to that on which primer (a) is based, but lacking the mutation. Thus, genetic material containing the non-mutated region will be amplified in the presence of primers (b) and (c). Genetic material homozygous for the wild type gene will thus provide amplified products in the presence of primers (b) and (c). Genetic material homozygous for the mutated gene will thus provide amplified products in the presence of primers (a) and (b). Heterozygous genetic material will provide amplified products in both cases.
The invention provides purified proteins having the biological activity of myostatin. The terms “isolated” and “purified” each refer to a protein substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. In certain preferred embodiments, the protein having biological activity of myostatin comprises an amino acid sequence identified as SEQ ID NO:2. Furthermore, proteins having biological activity of myostatin that are encoded by nucleic acids which hybridize under stringent conditions, as discussed above, to a nucleic acid comprising a nucleotide sequence identified as SEQ ID NO:1 or SEQ ID NO:7 are encompassed by the invention. Proteins of the invention having myostatin activity can be obtained by expression in a suitable host cell using techniques known in the art. Suitable host cells include prokaryotic or eukaryotic organisms or cell lines, for example, yeast, E. coil, insect cells and COS 1 cells. The recombinant expression vectors of the invention, described above, can be used to express a protein having myostatin activity in a host cell in order to isolate the protein. The invention provides a method of preparing a purified protein of the invention comprising introducing into a host cell a recombinant nucleic acid encoding the protein, allowing the protein to be expressed in the host cell and isolating and purifying the protein. Preferably, the recombinant nucleic acid is a recombinant expression vector. Proteins can be isolated from a host cell expressing the protein and purified according to standard procedures of the art, including ammonium sulfate precipitation, column chromatography (e.g. ion exchange, gel filtration, affinity chromatography, etc.), electrophoresis, and ultimately, crystallization (see generally, “Enzyme Purification and Related Techniques”, Methods in Enzymology, 22, 233-577 (1971)).
Alternatively, the protein or parts thereof can be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964), or synthesis in homogeneous solution (Houbenwcyl, 1987).
The protein of the invention, or portions thereof, can be used to prepare antibodies specific for the proteins. Antibodies can be prepared which bind to a distinct epitope in an unconserved region of a particular protein. An unconserved region of the protein is one which does not have substantial sequence homology to other proteins, for example other members of the myostatin family or other members of the TGFβ superfamily. Conventional methods can be used to prepare the antibodies. For example, by using a peptide of a myostatin protein, polyclonal antisera or monoclonal antibodies can be made using standard methods. A mammal, (e.g. a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the peptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art. For example, the peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay can be used to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.
To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures, thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art. For example, the hybridoma technique originally developed by Kohler and Milstein (Kohler, 1975) as well as other techniques such as the human B-cell hybridoma technique (Kozbor, 1983), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, 1985), and screening of combinatorial antibody libraries (Huse, 1989). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide, and monoclonal antibodies isolated.
The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with a protein having the biological activity of myostatin, or a peptide fragment thereof. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab′)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments.
It is also known in the art to make chimeric antibody molecules with human constant regions. See, for example, Morrison et al., Takeda et al., Cabilly et al., Boss et al., Tanaguchi et al., Teng et al. (Morrison, 1985; Takeda, 1985; Cabilly; Boss; Tanaguchi; Teng, 1982), European Patent Publication 0173494, United Kingdom Patent GB 2177096B, PCT Publication W092/06193 and EP 0239400. It is expected that such chimeric antibodies would be less immunogenic in a human subject than the corresponding non-chimeric antibody.
Another method of generating specific antibodies, or antibody fragments, reactive against protein having the biological activity of a myostatin protein, or a peptide fragment thereof, is to screen expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria, with peptides produced from the nucleic acid molecules of the present invention. For example, complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries. See for example Ward et al., Huse et al., and McCafferty et al. (Ward, 1989; Huse, 1989; McCafferty, 1990). Screening such libraries with, for example, a myostatin protein can identify immunoglobulin fragments reactive with myostatin. Alternatively, the SCID-hu mouse developed by Genpharm can be used to produce antibodies, or fragments thereof.
The polyclonal, monoclonal or chimeric monoclonal antibodies can be sued to detect the protein of the invention, portions thereof or closely related isoforms in various biological materials, for example they can be used in an ELISA, radioimmunoassay or histochemical tests. Thus, the antibodies can be used to quantify the amount of a myostatin protein of the invention, portions thereof or closely related isoforms in a sample in order to determine the role of myostatin proteins in particular cellular events or pathological states. Using methods described hereinbefore, polyclonal, monoclonal antibodies, or chimeric monoclonal antibodies can be raised to nonconserved regions of myostatin and used to distinguish a particular myostatin from other proteins.
The polyclonal or monoclonal antibodies can be coupled to a detectable substance or reporter system. The term “coupled” is used to mean that the detectable substance is physically linked to the antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 125I; 131I, 35S and 3H. In a preferred embodiment, the reporter system allows quantitation of the amount of protein (antigen) present.
Such an antibody-linked reporter system could be used in a method for determining whether a fluid or tissue sample of a subject contains a deficient amount or an excessive amount of the protein. Given a normal threshold concentration of such a protein for a given type of subject, test kits could be thus developed.
The present invention allows the skilled artisan to prepare bi-specific antibodies and tetrameric antibody complexes. Bi-specific antibodies can be prepared by forming hybrid hybridomas (Staerz, 1986 a & b).
Compositions of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. By “biologically compatible from suitable for administration in vivo” is meant a form of the composition to be administered in which any toxic effects are outweighed by the therapeutic effects of the composition. The term “subject” is intended to include living organisms in which a desired therapeutic response can be elicited, e.g. mammals. Examples of subjects include cattle, human, dogs, cats, mice, rats and transgenic species thereof. Administration of a therapeutically active amount of the therapeutic compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a compound that inhibits the biological activity of myostatin protein may vary according to factors such as the age, sex, and weight of the individual, as well as target tissue and mode of delivery. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
In the 2005 publication by Pirottin et al, entitled “Transgenic Engineering of Male-Specific Muscular Hypertrophy” (PNAS, May 3, 2005, vol. 102, no. 18, Pp. 6413-6418) the authors set forth a proof of principle relating to the use of a two-step procedure involving insertional gene targeting and recombinase-mediated cassette exchange in embryonic stem cells specific to transgenic mice. Pirottin et al showed that male mice produced in accordance with the disclosed methodology were characterized by a 5-20% increase in skeletal muscle mass. This led the authors to theorize that such methodology might be applicable to an efficient cattle production system wherein superior beef production and dairy abilities could be realized.
Given that embryonic stem cells are not available for cattle lines, the instant inventors have herein devised an alternative method for producing a genetically modified cattle production system wherein the phenotype exhibits the desirable attributes suggested by Pirottin et al.
By utilizing a somatic cell line, e.g. a fetal fibroblast cell line, in combination with a process for nuclear transfer, the instant inventors have now devised a process which should yield a transgenic line of cattle displaying male-specific muscular hypertrophy.
As illustrated in the following examples, a methodology is suggested for enabling the production of a transgenic bovine line having both male-specific muscular hypertrophy and enhanced dairy production abilities.
The instant inventors are interested in creating transgenic cattle which produce both milk and meat efficiently.
Intensive breeding programs implemented over the last 50 years have created cattle breeds that are highly specialized in either milk production (e.g. Holstein-Friesian and Jersey) or meat production (e.g. Angus, Hereford, Charolais, Piedmontese, and Belgian Blue). Physiological antagonisms have indeed precluded combining superior abilities for both milk and meat production in the same animal. Despite its effectiveness, the resulting production system can be considered suboptimal because of poor carcass and milk yield of beef and diary cattle, respectively.
The instant inventors envisioned a more efficient alternative based on specialization by sex within the same population: a breed in which cows would be of dairy type and bulls would be of beef type. To achieve this goal the instant inventors proposed to use genetic engineering techniques to target trans-inactivators of myostatin on the Y chromosome. In this way, males are predicted to exhibit muscular hypertrophy akin to “double-muscling”, whereas females will be non-transgenic and fully express their dairy potential.
In order to prove the feasibility of their concept in cattle, the instant inventors generated two transgenic lines of mice in which only the males express a myostatin trans-inactivator in skeletal muscle and consequently show an increase in individual muscles ranging from 5% to 20%.
Applicants note that the experimental design used to produce the described transgenic mice was approved by the ethics committee of the Faculty of Veterinary Medicine, University of Liège.
The instant inventors used a two-step procedure involving insertional gene targeting and recombinase-mediated cassette exchange in embryonic stem cells (ES cells) to produce transgenic mice.
The expression of the murine MSTN “latency-associated peptide” (LAP) or propeptide as a dominant-negative means to repress endogenous myostatin activity (Lee & McPherron PNAS USA 98:9306-9311 2001; Yang et al. Mol. Reprod. Dev. 60:351-361 2001; Thies et al. Growth Factors 18:251-259 2001; Hills et al. Journal of Biological Chemistry 277:40735-40741 2002; Wolfman et al. PNAS USA 100:15842-15846 2003). The testis-specific protein Y-encoded (TSPY) pseudogene was chosen as a targeting site on the murine Y chromosome, since contrary to other mammalian species where TSPY genes are multi-copy, the mouse TSPY is single-copy and non-functional despite being transcribed (Mazeyrat et al. Human Molecular Genetics 7:557-562 1998; Vogel et al. Chromosome Research 6:35-40 1998). As a consequence, the murine TSPy locus is predicted to be non-essential but transcriptionally competent. After Rohozinski et al. (Genesis 32:1-7 2002) the instant inventors chose insertional targeting rather than the usual replacement strategy (which has never been successfully applied to the murine Y chromosome) to insert a cassette containing a positive (neo) and a negative (HSV-tk) selectable marker flanked by heterologous lox sites into the murine Y chromosome. In a second stage, the marker cassette would then be exchanged through cre-mediated recombination with a cassette coding for a myostatin trans-repressor under the dependence of a strong skeletal muscle-specific promoter.
Construction of the Insertional Targeting Vectors pPNYdloxUP and pPNTdloxTSPY
Two adaptors containing (i) a loxP and a SalI site and (ii) a lox2272, a PacI, and a BamHI site were ligated through their shared AflII sticky ends into a 99-bp fragment with XbaI and EcoRI overhangs, which was directionally cloned in the corresponding restriction sites of the pPNT vector to yield the pPNTdlox vector. Homology arms corresponding to nt 31165-39425 [upstream(UP)] and nt 50690-57331 (TSPY) of sequence AC069015 (encompassing the murine TSPY gene) were amplified by using the Expand Long Template PCR system (Roche, Basel, Switzerland) from R1 genomic DNA with primers containing SalI and BamHI sites, respectively, at their extremities. This approach allowed convenient cloning of the PCR products in th pPNTdlox vector to yield the pPNTdloxUP and pPNTdloxTSPY plasmids. Approximately 300-bp gaps were introduced by digestion with Sad (pPNTloxUP) and BbvcI (pPNTdloxTSPY) followed by religation. An adaptor containing unique PmeI and AscI sites was introduced in the Sad site of the pPNTloxdUP. The gapped PPNTdloxUP and pPNTdloxTSPY vectors were completely sequenced before use.
Homologous recombination on the murine Y chromosome was successful using these insertional target vectors.
Two distinct insertional targeting vectors were generated by cloning (I) an 8.26-kb homology arm located 13.55 kb upstream of the TSPY pseudogene (pPNTdloxUP) and (ii) a 6.64-kb homology arm spanning the TSPY pseudogene (pPNTdloxTSPY), flanked by heterologous lox sites (loxP and lox 2272; Lee et al. Gene 216:55-65 1998 and Kolb A. F. Analytical Biochemistry 290:260-271 2001), in the pPNT vector providing the neo and HSV-tk cassettes (Tybulewicz et al. Cell 65:1153-1163 1991). The homology arms were obtained by long-template PCR from genomic DNA extracted from R1 ES cells. To enhance targeting efficiency and facilitate screening, 376- and 314-bp gaps (leaving unique AcsI and BbvcI restriction sites for linearization before electroporation) were generated in pPNTdloxUP and pPNYdloxTSPY, respectively. Gene targeting was performed in R1 ES cells by using standard procedures (Nagy et al. PNAS USA 90:8424-8428 1993 and Torres et al. Laboratory Protocols for Conditional Gene Targeting (Oxford University Press, New York) 1997). G418-resistant colonies (677 for pPNTdloxUP and 592 for pPNTloxdTSPY) were screened for successful insertion by using (I) PCR assays based on the use of vector-specific primers combined with gap-specific primers, followed by (ii) Southern blotting with a HSV-tk-specific probe and restriction enzymes cutting in the gap (pPNTdloxUP and pPNTdloxTSPY) and vector (PPNTdloxUP) and (iii) fluorescence in situ hybridization (FISH) by using a pPNT probe and a Y chromosome painting probe. For each construct, one properly targeted clone with euploid karyotype: RI-UP-neotk and RI-TSPY-neotk.
The targeting vectors were linearized with either AscI (pPNTdloxUP) or BbvcI (pPNTdloxTSPY), and 20 μg of resulting products was used to electroporate 107 R1 ES cells with the addition of 25 μg/ml spermidine in the electroporation medium. Positive selection was performed by using G418 (Invitrogen) at 300 μg/ml. After picking and replica plating, colonies having undergone the expected targeting event were identified by performing PCRs with primers located in the gap and selectable markers (neo and HSV-tk). At least two PCRs were performed for each construct, exploring the right and left boundaries of the integration site, respectively. The PCRs were carried out by using the Expand Long Template PCR system. Colonies that appeared positive after PCR screening were further analyzed by Southern blotting. DNA (7.5 μg) was digested with NdeI (pPNTdloxUP) or KpnI (pPNTdloxTSPY) and electrophoresed in a 1% agarose gel before blotting on a nylon membrane by using a standard alkali transfer procedure. The filter was hybridized to a 1,154-bp tk probe excised by BamHI-XbaI digestion from the pcDNA3hsvTK vector (coutesy of F. Princen, University of Liège) according to the manufacturer's instructions (Amersham Pharmacia). Finally, colonies positive by Southern blotting were analyzed by FISH. ES cell metaphase spreads were obtained by following standard procedures (Nagy et al. Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, N.Y.), third edition, 2003). The slides were treated with ribonuclease A and pepsin and fixed with 4% paraformaldehyde. Hybridization was performed at 37° C. in 2×Ssc buffer (1×SSC=0.15M sodium chloride/0.015M sodium citrate, pH 7) containing 50% formamide and 12.5% dextran sulfate. The probes were the flurescein-labeled pPNT plasmid and a Cy3-labeled murine Y chromosome painting probe (Cambio, Cambridge, U.K). The fluorescein signal was amplified by using the Tyramide Signal
Amplification System (NEN/PerkinElmer), and the slides were counterstained with DAPI before microscopic examination.
The R1-UP-neotk construct is shown in
The R1-TSPY-neotk construct is shown in
Southern blotting are shown. The negative clone (Neg. clone) represents a clone that has not undergone the proper gene targeting. The arrows point to the bands of expected size. Most clones that proved positive by PCR appeared to have multiple integrations of the transgene in an autosomal locus, explaining the multiple bands observed for the negative clones by Southern blotting.
Construction of the mDAFdloxLAP Vector
Adaptors containing loxP and lox2272 sites were cloned in the HindIII and EagI restriction sites located, respectively, upstream of the MLC1F promoter (myosin light chain, MLC) and downstream of MLC1/3E enhancer in the mDAF vector (Rosenthal et al. PNAS USA 86:7780-7784 1989). Proper orientation of the lox sites for compatibility with the pPNTdlox vector was verified by sequencing. The MSTN LAP-encoding sequence was obtained by RT-PCR from total RNA extracted from skeletal muscle of 2-month old mice by using TRIzol (Invitrogen). First-strand cDNA synthesis was carried out in a reaction volume of 20 μl starting from 2 μg of total RNA by using an oligo(dT)16as a primer and PowerScript reverse transcriptase (BD Biosciences/Clontech). RT-PCR was performed by using MSTN LAP-specific primers, including either an EcoRI tail or a SmaI tail. The RT-PCR product was digested with EcoRI and SmaI and cloned in the corresponding sites of the mDAFdlox vector. The completed mDAFdloxLAP vector was entirely sequenced before use.
Three million cells of the RI-UP-neotk and RI-TSPY-neotk ES cell clones were coelectroporated with 25 μg of mDAFdloxLAP and 50 μg of pMCcre plasmid in a buffer containing 25μg/ml spermidine. Gancyclovir-resistant clones (2 μM) were picked and replica-plated. Screening for the expected RMCE event was achieved by PCR with primers located in the UP and TSPY homology arms, the MLC1F promoter (PCR “A”), and MLC1/3E enhancer (PCR “B”). Clones that were positive by PCR were further analyzed by Southern blotting with HindIII restriction enzyme and the MSTN LAP as a probe. The MSTN LAP probe was obtained by PCR amplification of a 850-bp fragment from mDAFdloxLAP.
The MSTN trans-inactivator (LAP) was successfully integrated on the murine Y chromosome by means of RMCE.
The cDNA sequence coding for the murine MSTN LAP was obtained by RT-PCR from total skeletal muscle RNA and cloned into the mDAFdlox plasmid, properly placed under the dependence of the rat MLC1F promoter and 1/3 enhancer for expression in skeletal muscle. The mDAFdlox plasmid corresponds to the mDAF plasmid (Nagy et al. Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, N.Y.), third edition, 2003) in which a 1oxP sequence upstream of the MLC1F promoter and a lox2272 sequence downstream of the MLC1/3 enhancer were inserted. Clones RI-UP-neotk and RI-TSPY-neotk were coelectroporated with the mDAFdloxLAP and pMC-cre plasmids, the later encoding the cre recombinase under the dependence of a tk promoter (Gu et al. Cell 73:1155-1164 1993). Gancyclovir-resistant clones were screened for correct RMCE by (i) PCR assays with UP/TSYP- and MLC-specific primers followed by (ii) Southern blotting with a MSTN LAP probe. Ten RI-UP-LAP 1-10 clones and four RI-TSPY-LAP 1-4 clones having undergone proper RMCE were identified.
C57BL/6J blastocyts (3.5 days old) were harvested and microinjected with targeted ES cells as described in Nagy et al. (Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainview, N.Y.), third edition, 2003). Uterine transfer was performed the same day in CD1 pseudopregnant mothers by using standard procedures also described in Nagy et al. Individuals carrying the transgene were identified by using a multiplex PCR assay, allowing for the simultaneous amplification of an endogenous MSTN exon 1 fragment (230bp) and a transgene-specific fragment (450 bp) spanning the junction between the MLC1F promoter and MSTN LAP sequence. The ΔMCHR1 allele in the BC-CONT line (CONT, control) was detected by using a multiplex PCR generating a 450- and 700-bp fragment for the knockout and wild-type alleles, respectively.
Four of the R1-UP-LAP clones and all R1-TSPY-LAP clones were used for microinjection into recipient C57BL/6J blastocysts, followed by reimplantation in CD1 foster mothers (Nagy et al.
Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Plainveiw, N.Y.), third edition, 2003). Thirteen chimeric males (of which 6 were 100% agouti) and one chimeric females were obtained from R1-UP-LAP clones, whereas a single, 100% agouti chimeric male was obtained from a R1-TSPY-LAP clone. The seven 100% agouti chimeric males were mated to C57BL/6J females to yield an F1 generation. 110 males and 90 females (P=0.016) were obtained from three of the six R1-UP-LAP-derived males and 69 males and 47 females (P=0.04) were obtained from the unique R1-TSPY-LAP-derived chimera. As expected, all F1 males were shown by a PCR assay to carry the transgene, whereas none of the females did, thereby confirming the Y-specific integration and germ-line transmission of both UP-LAP and TSPY-LAP transgenes.
Total RNA was extracted from muscle and non-muscle tissue by using TRIzol (Invitrogen). Twenty micrograms of total RNA was denatured in formaldehyde load dye (Ambion, Austin, Tex.), electrophoresed on a Reliant Gel System (Cambrex, Rockland, Me.) in NorthernMax Mops gel running buffer (Ambion), and blotted on a positively charged nylon membrane (Amersham Pharmacia) by capillary transfer with 10 mM NaOH in 5×SSC buffer. The membrane was then hybridized overnight with 100 ng of a simian virus 40 (SV40) probe in ULTRAhyb hybridization buffer (Ambion) and washed in 0.1×SSC and 0.1% SDS-containing buffer. The SV40 probe was PCR amplified from the mDAFdloxLAP construct and labeled with 32P dCTP (Amersham Pharmacia) by random prime labeling (Invitrogen). Membranes were exposed on Hyperfilm (Amersham Pharmacia).
Transgene expression was assayed by Northern blotting with a SV40 probe and total RNA extracted from skeletal muscle, heart, and liver of a 13-week old F1 male and female from each line. In both lines, transgene-specific transcripts were detected exclusively in male skeletal muscle. As expected, there was no expression of the transgene either in the liver or the heart.
In order to analyze the phenotypic effect of the male-specific MSTN trans-inactivator, four F1-UP-LAP males and two F1-TSPY-LAP males were mated with 129\SV females to produce backcross (BC) animals. To generate a control population (BC-CONT), three non-transgenic males, derived from [(ΔMCHR1)R1>C58BL/6J] chimeric males (Adamantidis et al. European Journal of Neuroscience 21(10):2837-2844 2005) mated to C57BL/6J females, were crossed with 129/SV females. 184 BC-UP-LAPs (87 males and 97 females, P=0.46), 218 BC-UP-LAPs (114 males and 104 females, P=0.50), and 154 BC-CONTs (60 males and 94 females, P=0.006) were produced.
Transgene expression in the BC animals was assayed in skeletal muscle (pectoralis, triceps brachialis, quadriceps femoris, and gastrocnemius), heart, liver, and kidney of a 13-week-old male and female for both BC-TSPY-LAP and BC-UP-LAP lines. As expected, transgene-specified transcripts were detected exclusively in skeletal muscle of males BC-TSPY-LAP and BC-UP-LAP animals. Transgene expression seemed stronger and characterized by an increasing rostro-caudal gradient in the BC-TSPY-LAP animals (
BC animals were reared for 10 weeks during which they were weighed weekly. Analyzing the growth curves by using a mixed model including sex, age (weeks 4-10), genotype (UP-LAP, TSPY-LAP, or CONT), sex by genotype interaction, and random individual effects indicated that transgene genotype (both UP-LAP and TSPY-LAP) had a significant (P=0.0004) positive effect on weight; however, the effect did not differ significantly (P=0.96) between males and females. This observation indicates that at least part of the effect on growth is independent of transgene expression, thus probably due to polygenic background effects. R1 ES cells are of 129/SV×129cX/SV geneotype (Threadgill et al. Mammalian Genome 8:390-393 1197), the different BC lines could exhibit phenotypic differences due to variable proportions of 129/SV and 129cX/SV genes.
Live weight was recorded at 4, 5, 6, 7, 8, 9 and 10 weeks of age. Animals were killed at 10 weeks and dissected. The weight of the carcass (skinned body minus head, tail, all internal organs and associated fat and connective tissue), “leg weight” (skinned leg cut at the knee and tarsus level), and weights of the dissected pectoralis, triceps brachialis, and quadriceps femoris muscles were determined.
Growth curves were analyzed with PROC MIXED procedure of the SAS package (SAS Institute, Cary, N.C.). A mixed model including sex, genotype, sex by genotype interaction as fixed effects was used, and a random individual effect accounting for the covariances between repeated measurements (Litell et al. Journal of Animal Science 76:1216-1231 1998) was also used. Relative muscle weights as well as myofiber diameter were analyzed separately for each sex by using the PROC GLM procedure of the SAS package and a model including genotype as a fixed effect.
In order to test for an effect of transgene expression on muscle mass, all BC animals at 10-weeks of age were killed and the carcass, the leg, and a series of individual muscles were weighed. To correct for the differences in live weight observed between lines and individuals, carcass, leg and muscle weights were divided by live weight at slaughter. When analyzing males, both transgenic lines (BC-UP-LAP and BC-TSPY-LAP) exhibited highly significant increases in relative carcass, leg, and individual muscle weights when compared with the control line (BC-CONT). Carcass and leg weights were increased by ≈5%, triceps brachialis weight was increased by ≈10%, and quadriceps femoris weight was increased between ≈15% (BC-UP-LAP) and ≈20% (BC-TSPY-LAP); Tables 4A-B. When comparing females, on the contrary, there was no evidence at all for an effect of genotype (UP-LAP, TSPY-LAP, or CONT) on normalized carcass, leg, or individual muscle weights; Tables 4A-B. These results strongly suggest that the effects observed in the males are caused by the transgenes. The stronger effect in quadriceps femoris (hind legs) when compared with triceps brachialis (front legs) and pectoralis in both lines supports the occurrence of a rostro-caudal gradient and corroborates the Northern blot results in the BC-TSPY-LAP line. Weights of the triceps brachialis and quadriceps femoris were slightly higher in the BC-TSPY-LAP than in the BC-UP-LAP males (P=0.06 and 0.03, respectively), suggesting that the transgenic effect is larger in the former, again corroborating the findings of the Northern blots.
The increase in weight observed for UP-LAP and TSPY-LAP females (
Transgenic expression of the MSTN propeptide has been shown to cause an increase in myofiber diameter (Lee & McPherron PNAS USA 98:9306-9311 2001 and Yang et al. Mol. Reprod. Dev. 60:351-361 2001). To test whether a similar myofiber hypertrophy would have been induced by the transgene in BC-UP-LAP and BC-TSPY-LAP males, histological examinations of transverse sections of the quadriceps femoris was performed. Five 10-week-old males and seven females for each of the three lines (BC-UP-LAP, BC-TSPY-LAP, and BC-CONT) were analyzed. To ensure representativeness, animals with a weight at slaughter within 0.5 g of the mean of their sex-genotype class were selected. The diameter of all myofibers within 10 consistently positioned ×40 microscopic fields for an average of 158 myofibers per individual was determined.
Ten-week-old mice were killed, and their quadriceps femoris were dissected and fixed in 4% buffered formaldehyde. Muscles were cut transversally at the midpoint and embedded in paraffin. Four-micrometer-wide transverse sections were made from the widest part of the muscle and stained with antibodies against collagen IV to facilitate visualization of individual fibers. Antigen was demasked by pepsin treatment for 60 minutes, and slides were incubated two times (1:5,000 and 1:500) with anti-collagen IV rabbit polyclonal antibody AB748 (Chemicon, Temecula, Calif.). For each muscle section, 10 photographs were taken at ×40 magnification, these photographs being evenly dispersed throughout the section and consistently positioned across individuals. All of the entire myofibers within the microscopic field were measured by using ANALYSIS 3.2 image analysis software (Soft Imaging System, Münster, Germany), and fiber diameter was considered to be the diameter of the largest circle that could be placed within each myofiber.
The production of the transgenic mice described above demonstrates that it is feasible to engineer strains of mammals in which only males express a muscular hypertrophy as a result of the expression of trans-inactivators of the myostatin gene from a transgene integrated on the Y chromosome.
These methods can be optimized for use in cattle.
Currently, embryonic stem cells (ES cells) are available only for application in mice. However, several protocols, for example, nuclear transfer (using somatic cells) useful for successful production of transgenic calves are known in the art (Kuroiwa et al. Nature Genetics 36(7):775-780 2004; Sullivan et al., Biological Reproduction 70:146-153 2004; Kuroiwa et al. Nature Biotechnology 20:889-894 2002; Cibelli et al. Science 280:1256-1258 1998 and U.S. Pat. No. 5,633,076).
The method would involve obtaining a somatic cell, preferably, but not limited to, a fetal fibroblast, introducing a transgene of interest to the somatic cell, introducing the nucleus of the transformed somatic cell to an enucleated oocyte, cultivating the oocyte to obtain an embryo and inserting the embryo into the uterus of a foster mother. The method taught by Kuroiwa et al. 2004, a sequential gene targeting strategy for primary somatic cells, is particularly appropriate.
The TSPY gene used to generate the transgenic mice is not a suitable target site on the bovine Y chromosome as this gene is functional in cattle. Finding alternative target sites, i.e. genes that are transcribed in cattle but have no function, would not present any difficulties for a skilled artisan as a substantial portion of the bovine Y chromosome is currently being sequenced.
In order to identify suitable target sites on the bovine Y chromosome, the instant inventors have isolated clones from a bovine BAC library containing inserts that originate from the bovine Y chromosome. More specifically, a BAC clone that spans the psuedo-autosomal boundary on the Y chromosome has been sequenced and annotated. The 190 kb insert is identified as SEQ ID NO:80.
Furthermore, additional Y-specific sequences are known and available to the public.
In some cattle breeds, particularly in the BBB, the “double-muscling” phenotype is associated with a high incidence of dystocia, leading to a nearly systematic reliance on cesarean section in some countries. This major drawback has limited the dissemination of the BBB to most countries. The high incidence of dystocia in BBB is due to (i) the extreme muscular hypertrophy characterizing BBB that results from the combined effect of loss-of-function mutation in the myostatin gene and additional “polygenic” effects and (ii) the extreme muscularity of the calf, and also the cow, resulting in a narrowed pelvic channel.
The instant invention remedies this drawback (calving difficulties) because (i) the muscular hypertrophy will be less extreme than, for example, in the BBB, and (ii) the cows will be non-transgenic, and hence, of the dairy type. In addition, one can envisage delaying expression of the myostatin trans-inactivators in order to obtain a postnatal expression of the muscular hypertrophy. Such delayed expression could be achieved by using promoters that are becoming active only in later stages of development or that are inducible through exogenous means. The instant inventors have demonstrated the effectiveness of delayed myostatin invalidation in obtaining late-onset muscular hypertrophy by using cre-loxP-mediated conditional myostatin invalidation (Grobet et al. Genesis 35:227-238 2003).
In summary, the instant inventors, using a two-step procedure involving gene targeting and recombinase-mediated cassette exchange in ES cells, have produced two lines of transgenic mice expressing a dominant-negative latency-associated myostatin propeptide under control of the myosin light chain 1F promoter and 1/3 enhancer from the TSPY locus on the Y chromosome. Males of the corresponding lines are characterized by a 5-20% increase in skeletal muscle mass. This invention enables a more efficient cattle production system combining superior beef production abilities for bulls and diary abilities for cows.
All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. It is to be understood that while a certain form of the invention is illustrated, it is not to be limited to the specific form or arrangement herein described and shown. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objectives and obtain the ends and advantages mentioned, as well as those inherent therein. The transgenic animals, oligonucleotides, peptides, polypeptides, biologically related compounds, methods, procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.
Particulars of references cited are given below. All of the listed references are incorporated herein by reference.
G. Woodfall).
This application is a continuation of application Ser. No. 11/416,780, filed on May 2, 2006, which is a continuation-in-part of application Ser. No. 10/251,115, filed on Sep. 20, 2002, now abandoned, which is a continuation of application Ser. No. 09/007,761, filed on Jan. 15, 1998, now abandoned, which is a continuation-in-part of application Ser. No. 08/891,789, filed on Jul. 14, 1997, now U.S. Pat. No. 6,103,466, the contents of which are each herein incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11416780 | May 2006 | US |
| Child | 12322075 | US | |
| Parent | 09007761 | Jan 1998 | US |
| Child | 10251115 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10251115 | Sep 2002 | US |
| Child | 11416780 | US | |
| Parent | 08891789 | Jul 1997 | US |
| Child | 09007761 | US |
