Patent Application
20070292952
Fish and shellfish farming significantly contribute to the global food supply and is a source of a major economic activity in developing nations (FAO Fisheries department, 2000). As the supply of food fish and shellfish from capture fisheries declines globally, there is an urgent need to enhance aquaculture production. The development of aquaculture in a sustainable manner faces a number of challenges. Among them diseases caused by diverse etiologic agents is of particular importance. Disease outbreaks and emergence of new pathogens poses a major challenge for sustainable development in aquaculture. A case in point is shrimp aquaculture.
During the past decade, shrimp (Penaeus sp.) farming has evolved from subsistence level farming to a major worldwide industry providing jobs to millions of people both directly and indirectly, particularly in the coastal areas of the developing nations in the Asia and the Central and South Americas. Among the challenges facing shrimp farming globally, economic losses from diseases caused by viruses are a major concern. Since the first report of a viral disease in early 1970s, more than 20 viruses have been reported that infect shrimp (Lightner, 1996, Rev. Sci. Tech. Off. Int. Epiz. 15:579-601). This list is growing rapidly. Many of shrimp viruses have caused serious epizootics in penaeid shrimp resulting in significant economic losses to commercial shrimp farmers and potentially affecting wild crustacean populations adversely. The four most important viruses of penaeid shrimp are white spot syndrome virus (WSSV), yellowhead virus (YHV), Taura syndrome virus (TSV) and the infectious hypodermal and hematopoietic necrosis virus (IHHNV). WSSV, YHV, and, more recently, TSV have caused serious epizootics in the Eastern Hemisphere; whereas, WSSV, TSV and IHHNV related economic losses have occurred in the Western Hemisphere (Dhar et al., 2004, Adv. Virus Res., In press; Tu et al., 1999, Dis. Aquat. Org. 38:159-161; Lightner et al., 1996, Rev. Sci. Tech. Off. Int. Epiz. 16:146-160). Considerable progress has been made in developing detection methods and characterizing these viral pathogens at molecular level over the last few years. However, information on the role of the virally encoded proteins in viral pathogenesis and the genes involved in host anti-viral response remains largely unknown. This is primarily because of: (1) a lack of a suitable transient and transfection vectors for shrimp and other crustaceans and (2) a lack of a permanent cell line for shrimp and any other crustaceans.
The instant invention addresses these issues by developing expression vectors for transient expression of foreign genes, and for transfection of shrimp primary cell lines with foreign genes or modifiers of endogenous genes. These vectors could be used (1) to express recombinant protein(s) with therapeutic potential using shrimp or other crustacean host, (2) to express host gene or foreign gene in excess to determine their role in growth, development, and or disease resistance using shrimp or other crustacean host, (3) to develop a transgenic shrimp or other crustaceans, and (4) to study the role of virally encoded protein in viral pathogenesis in vitro and in vivo. These techniques will develop the tools needed to modify primary cell cultures to enable the development of unregulated growth necessary for immortalized cell lines.
The inventors have discovered a number of important tools that will enable the manipulation of animal genes both transiently and permanently in both tissue culture and live animals. In general, the invention harnesses elements from shrimp viruses that through bioinformatics evaluation have been shown to have homology to enhancer or regulatory elements in other species. The inventors have demonstrated specific vectors that can be developed from these elements and methods of their use in the instant invention that will allow the improvement of animal health as well as diagnostic uses thereof. Specifically the invention relates to improvement of crustacean and especially shrimp health in relation to virally transmitted disease. The invention also provides tools for drug development and diagnostics of the same.
It is an object of the invention to provide a transient expression vector for the expression of a foreign gene or genes in Penaeid shrimp tissues in vitro or in vivo.
It is a further object of the invention to provide a transient expression vector for the expression of a foreign gene or genes in bacteria and yeast.
It is an object of the invention to provide a transfection vector for the expression of a foreign gene or genes in Penaeid shrimp tissue in vitro or in vivo.
It is an object of the invention to provide a transfection vector for developing transgenic shrimp expressing a foreign gene or shrimp gene or any combination thereof.
It is an object of the invention to provide a transient and a transfection vector for developing a permanent cell line for shrimp, crustaceans, and mollusks by over expressing host telomerase gene or introduction of a heterologous telomerase gene.
It is an object of the invention to provide a transfection vector for developing transgenic fish, shellfish or insects expressing a foreign gene or genes. Such a vector would have a broad host range that may be applicable to other animals as an expression system.
It is an object of the invention to provide methods of transient expression in crustaceans, fish, shellfish, insects, or other cell culture based on the vectors developed herein.
It is an object of the invention to provide methods for transfection in crustaceans, fish, shellfish, insects, or other cell culture based on the vectors developed herein.
It is an object of the invention to provide methods of protecting shrimp from WSSV, IHHNV, TSV, YHV and other viral, bacterial and fungal diseases by over expressing antiviral/antimicrobial factors using the transfection vector.
It is an object of the invention to provide methods of protecting shrimp from WSSV, IHHNV, TSV, YHV and other viral, bacteria, and fungal diseases by over expressing pathogen-encoded protein (e.g., capsid protein) using the transfection vector.
It is an object of the invention to provide methods of protecting fish and shellfish from viral, bacterial, fungal and or other microbial infection by over-expressing antiviral/antimicrobial factors using the transfection vector for delivery of the therapeutic effect.
It is an object of the invention to provide methods of protecting fish and shellfish from viral, bacterial, fungal and or other microbial infection by over expressing pathogen-encoded protein or proteins (e.g., capsid protein, enzyme, receptor, recognition sequence) using the transfection vector.
Therefore, the invention provides a composition for developing a transient or transfection vector using sequences of shrimp virus and shrimp cellular gene as well as the methods for using such vectors for manipulation of cells and diagnostic applications. Such vectors could be used for the expression of recombinant protein in vitro or in vivo in shrimp or other crustaceans, fish, insects, and other animals.
The invention relates to compositions and methods that can be used to express heterologous nucleic acids and proteins in cells of marine creatures, including crustaceans such as shrimp, lobster, crayfish, and crabs, and further including non-crustacean arthropods and other marine animals such as fish and mollusks.
The invention relates to a non-naturally-occurring nucleic acid construct that includes at least a viral promoter region and an insect picomavirus (IPV) internal ribosome entry site (IRES). One or both of the promoter and the IRES can be linked with one or more cistrons, such as an intron-free open reading frame (ORF). Significantly, it has been discovered that use of a virus promoter and an IRES from an IPV renders the construct capable of expressing in a crustacean cell RNA or protein encoded by a nucleic acid introduced into the cell as part of the construct. Such constructs can be used to produce one or more nucleic acids in a host crustacean cell. By way of example, an mRNA encoding a protein of interest can be produced, as can an RNA that binds with a specific endogenously-produced nucleic acid of the cell. Further by way of example, a protein encoded by a portion of the construct can produced in a crustacean cell by incorporating into the cell a construct in which the portion is operably linked to a viral promoter to generate RNA that includes both the portion and an IPV IRES, which can be translated by the host cell to produce the protein.
The identities of the virus from which the promoter and the IPV from which the IRES are derived is not critical. Preferably, the promoter and the IRES are derived from the same IPV, but elements from distinct viruses can be used as well. The IPV(s) can be substantially any IPV that is currently known or hereafter developed. A preferred type of IPV is the class of IPVs that are designated “cricket paralysis-like” picornaviruses, as described in the art (e.g., Christian et al., 1998, In: Miller et al., Eds., “The Insect Viruses” New York, Plenum Press, pp. 301-329; van Regenmortel et al., 2000, In: “Virus Taxonomy: The Classification and Nomenclature of Viruses. The Seventh Report of the International Committee on Taxonomy of Viruses” Academic Press, San Diego). It is recognized that it is not always possible to distinguish picornaviruses from other RNA-containing viruses which exhibit significant morphological, physical, and chemical similarity to picornaviruses. Thus, the art recognizes a group of “picorna-like” viruses, such as TSV of shrimp. As used herein, the term IPV includes both viruses that are capable of infecting hosts in phylum Arthropoda, regardless of whether they are designated “picornaviruses” or “picorna-like viruses.”
Knowledge of the identity of a host cell into which a nucleic acid construct of the type described herein will be incorporated can guide selection of an appropriate IPV. It has been discovered that promoters from substantially any virus (at least viruses suitable for any actual or anticipated host for the construct) and IRES' from substantially any IPV can be used in vectors intended for use in crustaceans. Nonetheless, it can be preferable to select a promoter, an IRES, or both, from a virus that is known to infect crustaceans of the same species as the crustacean from which the host cell was derived. Thus, for example, if a nucleic acid construct described herein is to be used to produce a protein upon incorporation into shrimp cells, it can be preferable to use a promoter and IRES from among those of known shrimp viruses, such as infectious hypodermal and hematopoietic necrosis virus (IHHNv), Taura syndrome virus (TSV), or white spot syndrome virus (WSSV).
The properties of many viral promoters are known, including multiple IPV promoters. Substantially any viral promoter can be used in the nucleic acid constructs described herein. Examples of suitable promoters include promoters from TSV, IHHNV, and WSSV (e.g., the P2 or P61 promoter of WSSV).
In many picomaviruses, especially including those having genes arranged in a single polycistronic unit, an IRES occurs in the 5′-untranslated region of the viral genome. In other picomaviruses, an IRES occurs in the intergenic region between two or more cistronic regions of the viral genome. Either type of IRES can be incorporated into the nucleic acid construct described herein.
The nucleic acid constructs described herein can be designed to express a product for a relatively short time or for a longer period, such as the lifetime of a host cell or organism, or even across generations of organisms. The association between the form of a nucleic acid construct (e.g., straight-chain nucleic acid, plasmid, or viral vector) and its duration in vivo are known and a skilled artisan can influence the duration of product production from the construct by selection of a suitable form of the construct.
Duration of product production can also be influenced by the properties of the genetic elements included in the construct. By way of example, the promoter that is used can be an inducible or repressible promoter, subject to regulation by its effectors. Similarly, it is known that promoters can induce transcription of an operably linked sequence at a high or low level, depending on the identity of the promoter and the host organism.
Residence in a host cell of a construct described herein can be influenced by the presence or absence of genetic elements in the construct. By way of example, if the construct in introduced into a host cell in the form of a plasmid, the plasmid (together with the construct) will be replicated if the plasmid includes an origin of replication site (ORS) that is operable in the host cell. It is known that ORS can induce maintenance of single, a few, or multiple copies of a plasmid in a cell, and a skilled artisan can select an appropriate ORS for a desired copy number for a desired host cell. Further by way of example, if the construct is included in a virus vector construct that is capable of replication and packaging (or can be induced to replicate and package), then maintenance or spread of the construct can be controlled using methods understood by a skilled artisan in this field.
One or more cistronic elements (e.g., an ORF) can be operably linked with the IRES of the construct, to thereby allow expression of the protein or protein subunit encoded by the cistron. The cistronic element should also be operably linked with the promoter, so as to enhance transcription of RNA encoding the cistron prior to translation brought about by introduction of a ribosome into the RNA by way of the IRES (or by a normal, mRNA cap-targeted ribosome or both). The cistron is preferably an ORF that is not interrupted by an intron, but one or more introns can be present so long as the host cell is able to remove the intron(s) to yield the construct-encoded product of interest.
A cistron of the construct can encode an RNA that has a physiological effect on the host cell (e.g., one that hybridizes specifically with a nucleic acid in the cell). The cistron can also encode a protein. For example, if the cell is a cell of a crustacean food product (e.g., a farm-raised shrimp), the protein can be one which enhances the human nutritional value, the health of the crustacean, or the disease resistance of the crustacean. Proteins which enhance the lifespan, physiology or rate of proliferation of an individual cell are further examples of proteins that can be expressed in a host cell from the nucleic acid construct described herein.
It is known that a protein or nucleic acid expressed in a cell can exert a direct physiological effect on a cell (e.g., a protein that catalyzes a chemical reaction or is a structural component of a cell or a nucleic acid that binds with a cellular nucleic acid in way that affects expression of the cellular nucleic acid). It is also known that a protein or nucleic acid expressed in a cell can exert a relatively indirect physiological effect on a cell (e.g., a protein that acts as a transcription factor or a nucleic acid that modulates the activity of a cellular nucleic acid polymerase). It is immaterial which of these (or other) types of products is produced from a nucleic acid construct described herein when the construct is introduced into a host cell. A skilled artisan can select an appropriate product based on the identity and biochemical properties of the host cell and the desired effect, and no more than ordinary experimentation is required to make a construct that produces the appropriate product. Such products can be products that are ordinarily produced in the host cell (e.g., at a different level in the presence and absence of the construct) or can be heterologous products (e.g., products unlike any produced by the host cell or an altered form of a homologous product).
A transposable element (TE) can be included in the construct if desired. A TE facilitates distribution of operably linked elements of the construct into the genome of the host cell. A TE can thereby incorporate a transgene from the construct described herein (transiently or relatively permanently, depending on the TE selected) into the host genome. A skilled artisan is able to operably link selected elements of the construct described herein with a TE with no more than routine experimentation. Examples of suitable TEs include those that are homologous to the Drosophila Penelope TE sequence reported in the literature. Other suitable TEs include IPV TEs, such as those of WSSV.
In an important embodiment, the nucleic acid construct described herein can be used to express a protein in a host cell, such as a crustacean cell. This is achieved by introducing into the host cell one or more copies of the nucleic acid construct described herein. The method by which the construct is introduced into the host cell is not critical, and a variety of methods of introducing a nucleic acid vector into a cell are known in the art. Examples of suitable methods of incorporating the construct into a host cell include use of a virus vector containing the construct, a ballistically administered particle coated with or containing the construct, and electroporesis of cells in the presence of a plasmid that includes the construct.
Definitions
In describing the present invention, the following terminology is used in accordance with the definitions set out below.
“Shrimp” refers to any of the group of crustaceans that are commonly cultured for aquaculture or captured in the wild fisheries. The term “shrimp” includes shrimp eggs, shrimp larvae, shrimp post-larvae and adult shrimp. The term “shrimp” and “prawn” will be used interchangeably throughout the specification. Shrimp can refer to but are not limited to, Penaeus shrimp and include the species Penaeus vannamei, Penaeus chinensis, Penaeus monodon, Penaeus stylirostris, Penaeus japonicus, Penaeus penicillatus, Penaeus merguiensis, Penaeus indicus, Penaeus subtilis, Penaeus paulensis, Penaeus setiferus, Penaeus brasiliensis, Penaeus duorarum, Penaeus occidentalis, Penaeus schmitti, Penaeus californiensis, Penaeus semisulcatus, Penaeus latisulcatus, Metapenaeus monoceros, Metapenaeus dobsoni, Metapenaeus affinis, and Metapenaeus brivicornis; and Litopenaeid shrimp (such as Litopenaeus vannamei, L. setiferus).
A “vector” comprises a nucleic acid, which can infect, transfect, and transiently or permanently transform or transfect a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. The vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and include both the expression and non-expression plasmids. Where a recombinant microorganism or cell culture is described as hosting an “expression vector” this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained in a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
The term “promoter” includes all sequences capable of driving transcription of a coding sequence in a cell, e.g., a plant cell, animal cell, bacterial cell, fungal cell, and yeast cell. Thus, promoters used in the constructs of the invention include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene or genes. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription. “Constitutive” promoters are those that drive expression continuously under most environmental conditions and states of development or cell differentiation. “Inducible” or “regulatable” promoters direct expression of the nucleic acid of the invention under the influence of environmental conditions or developmental conditions. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, drought, or the presence of light.
“Plasmids” can be commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. Equivalent plasmids to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.
“Transposable elements” (TEs) or “transposons” are a group of genetic elements that move from one locus to another by non-homologous recombination.
“Internal ribosome entry site” or “IRES” refers to a translation control element for cap independent mRNA translation. IRES' have been described in the art (e.g., see review by Hellen et al., 2001, Genes Develop. 15:1596-1612), and a skilled artisan is able to operably link an open reading frame or cistron with an IRES with no more than routine experimentation.
The term “gene” includes a nucleic acid sequence comprising a segment of DNA involved in producing a transcription product (e.g., a message), which in turn is translated to produce a polypeptide chain, or regulates gene transcription, reproduction or stability. Genes can include regions preceding and following the coding region, such as leader and trailer, promoters and enhancers, as well as, where applicable, intervening sequences (introns) between individual coding segments (exons).
The phrases “nucleic acid” or “nucleic acid sequence” includes oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin which may be single-stranded (ss) or double-stranded (ds) and may represent a sense (+) or antisense (−) strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs). The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones (see e.g., Mata, 1997, Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup, 1997, Biochemistry 36:8692-8698; Samstag, 1996, Antisense Nucl. Acid Drug Dev. 6:153-156).
“Amino acid” or “amino acid sequence” includes an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules. The terms “polypeptide” and “protein” include amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids. The term “polypeptide” also includes peptides and polypeptide fragments, motifs and the like. The term also includes glycosylated polypeptides. The peptides and polypeptides of the invention also include all “mimetic” and “peptidomimetic” forms, as described in further detail, below.
The term “isolated” includes a material removed from its original environment, e.g., the natural environment if it is naturally occurring. For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. As used herein, an isolated material or composition can also be a “purified” composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition. Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity. In alternative aspects, the invention provides nucleic acids, which have been purified, from genomic DNA or from other sequences in a library or other environment by at least one, two, three, four, five, or more orders of magnitude.
As used herein, the term “recombinant” can include nucleic acids adjacent to a “backbone” nucleic acid to which it is not adjacent in its natural environment. In one aspect, nucleic acids represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid “backbone molecules.” “Backbone molecules” according to the invention include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest. In one aspect, the (isolated, recombinant, enriched) nucleic acids represent 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the number of nucleic acid inserts in the population of recombinant backbone molecules. “Recombinant” polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein. “Synthetic” polypeptides or protein are those prepared by chemical synthesis, as described in further detail, below.
A promoter sequence can be “operably linked to” a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into MRNA, as discussed further, below.
“Primer” includes either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands, which may be chemically synthesized. Such synthetic oligonucleotides have no 5′ phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide can ligate to a fragment that has not been dephosphorylated.
All other terms are defined in the literature using Sambrook (Sambrook et al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor) as a guide.
Genome Organization of the TSV
The TSV genome is a single-stranded RNA of positive polarity with a 3′-poly(A) tail (Bonami et al., 1997, J. Gen. Virol. 78:313-319). The genome is 10205 nucleotides (nt) long with a 5′ untranslated region of 377 nt and a 3′ untranslated region of 226 nt (Mari et al. 2002). There are two open reading frames (ORFs) in the TSV genome. ORF1 is 6324 nt long, and encodes a 2107 amino acid (aa) polyprotein with a molecular mass of 234 kDa. ORF2 is 3036 nt long and encodes a 1011 aa polypeptide with a molecular mass of 112 kDa (Mari et al., 2002, J. Gen. Virol. 83:915-926; Robles-Sikisaka et al., 2001, Arch. Virol. 146:941-952). There is an intergenic region of 226 nt between the two ORFs. ORF1 encodes non-structural proteins (helicase, a protease and a RNA-dependent RNA polymerase, RdRp), and ORF2 encodes the virion structural proteins (
Comparison of TSV Genome Organization with Insect and Mammalian Picornaviruses.
Picornaviruses have been isolated from a wide range of insect species. Based on their biological, biophysical properties, and genome organization data these viruses were classified as a member of a newly designated group, “Cricket paralysis-like viruses” in Picornaviridae with CrPV as the type species of this group (Christian et al., 1998, In: Miller et al., Eds., “The Insect Viruses” New York, Plenum Press, pp. 301-329; van Regenmortel et al., 2000, In: “Virus Taxonomy: The Classification and Nomenclature of Viruses. The Seventh Report of the International Committee on Taxonomy of Viruses” Academic Press, San Diego). Genomes of a number of these viruses have now been sequenced. These include CrPV (GenBank accession number AF218039), DCV (AF014388), acute bee paralysis virus (ABPV, NC002548); black queen cell virus (BQCV, AF183905) and SBV (AF092924) of honeybees; RhPV (AF022937), PSIV (AB006531), TrV (AF178440), HiPV (AB017037); IFV (AB000906) and TSV of shrimp (AF277675). Among these viruses, the genome organizations of IFV and SBV were found to be similar to that of mammalian picornaviruses. They contain a single long ORF with the capsid proteins located at the N-terminal end and the non-structural proteins at the C terminal end. In contrast, the genomes of CrPV, DCV, RhPV, PSIV, HiPV, TrV, and TSV contain two long ORFs (ORF1 and ORF2) separated by an intergenic region. For all but RhPV, the length of the intergenic region varies from 171-207 nucleotides. The intergenic region of RhPV is 525 nucleotides long. The 5′ end of ORF1 contains the non-structural proteins and the 3′ end of ORF2 contains the capsid proteins (
The invention, as contemplated herein, is a composition comprising nucleotide sequence of an insect picornavirus (IPV) such as the Taura syndrome virus (TSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), or white spot syndrome virus (WSSV), and one or more cistronic elements (e.g., a shrimp gene sequence) operably linked there with. Alternatively, the invention provides methods of protection of animals, in particular crustaceans from disease and manipulation of animals, specifically crustaceans, in vitro and in vivo. The invention provides tools critical for the production of an immortalized shrimp cell line. The following examples describe some aspects of the invention and are used for exemplification purposes only and are not intended to limit the scope of the invention in any way.
Identification of Internal Ribosomal Entry Site (IRES) Elements in TSV and Insect Picorna-Like Viruses.
Insect picornaviruses with dicistronic genomes have two unique features. First, no sub-genomic RNA is produced for translation of the capsid proteins. Second, the coat protein cistron appears to lack an initiating methionine suggesting that the coat protein is translated through internal initiation mediated by an internal ribosomal entry site (IRES).
Functional IRES elements have been identified in the intergenic region of CrPV and PSIV (Wilson et al., 2000, Mol. Cell. Biol. 20:4990-4999; Sasaki et al., 1999, J. Virol. 73:1219-1226), and homologous sequences have been identified in TSV (Mari et al., 2002, J. Gen. Virol. 83:915-926,
Multiple alignments of the intergenic regions of insect picorna-like viruses are shown in
The nucleotide residues that constitute the stem loop structure right before the ORF2 is indicated in
In insect picorna-like viruses like DCV, it has been shown that structural proteins are produced in vast excess over non-structural proteins in virus-infected cells (Moore et al., 1980, J. Virol. 33:1-9; Moore et al., 1981, Arch. Virol. 68:1-8). This is in contrast to human picomavirus-infected cells, wherein approximately equimolar amounts of structural and non-structural proteins are produced (Ruckert, 1996, In Fields et al., Eds., “Field Virology” Lippincott-Raven, Philadelphia, Pa., pp. 609-654). The IRES-mediated translation of the coat proteins in insect picorna-like viruses with dicistronic genome, therefore, provides a mechanistic explanation for the abundance of structural compared to non-structural proteins in insect picorna-like virus infected cells. Insect picorna-like viruses with dicistronic genomes encode two distinct polyproteins: non-structural proteins encoded by the upstream ORF and the structural protein encoded by the downstream ORF. Translation of the two ORFs seems to be independently controlled. This is in contrast to the picomaviruses with single ORF genomes, which are translated into a single polyprotein and post-translationally processed to give rise to both structural and non-structural proteins (Ruckert, 1996, In Fields et al., Eds., “Field Virology” Lippincoft-Raven, Philadelphia, Pa., pp. 609-654).
All picomaviruses that infect mammalian, insect, or plant hosts, contain a 5′-untranslated region. Like the IRES element in the intergenic region of insect picoma-like viruses with dicistronic genome, the 5′ untranslated region of mammalian picornaviruses were shown to contain secondary structures that serve as an IRES element and thus, capable of cap-independent translation.
Genome Organization of IHHNV
The IHHNV genome contains a single stranded linear DNA of 4.1 kb (Bonami et al., 1990). Almost the entire IHHNV genome except the 5′-and 3′-terminal ends has been sequenced (Shike et al., 2000, Virology, 277:167-177). There are three large open reading frames (ORFs) in the IHHNV genome (
The left ORF most likely encodes for a major non-structural protein (NS-1), which contains replication initiation motifs, NTP-binding, and helicase domains. The right ORF encodes the capsid protein. The aa sequence of middle ORF does not show any similarity with database entries, and therefore, the putative function of this ORF is unknown. There are two potential promoters identified in IHHNV genome. One called P2 (based on map unit 2) is located upstream of the left ORF, and the other, called P61, is located upstream of right ORF (nucleotide position 2398-2448). Overall, the genome organization revealed that IHHNV is closely related to densoviruses of the genus Brevidensovirus in the family Parvoviridae (Shike et al., 2000, Virology, 277:167-177). A schematic representation of the genome organization of IHHNV genome is shown in
Amplification of IHHNV P61 Promoter by Polymerase Chain Reaction (PCR)
In order to evaluate the promoter activity of IHHNV P61 promoter, a 166 bp DNA flanking the P61 promoter was designed. The sequence of the primers were: IHHNP61F: 5′-GGTAC CTCCA GCTGA TGGTA AAGCT-3′ (SEQ ID NO: 19; nucleotide position 2346-2371 in AF273215) and IHHNP61R: 5′-TTCGT ATTCT TGGAA GAGTC CTAG3″ (SEQ ID NO: 20; nucleotide position 2488 in AF273215). The reaction mixture for the PCR included 1× Promega thermophilic DNA polymerase buffer, 2.0 millimolar MgCl2, 0.4 micromolar dNTPs, 0.8 micromolar of forward and reverse primer, 7.5 units of Promega Taq DNA polymerase, and 100 nanograms of total genomic DNA isolated from IHHNV-infected shrimp in a 25 microliter reaction volume. The thermal profile for the PCR was 95 degrees Celsius for 5 minutes followed by 40 cycles of 95 degrees Celsius 30 seconds, 52 degrees Celsius 30 seconds, and 72 degrees Celsius 1 minute before cooling at 4 degrees Celsius. PCR amplified DNA was run in a 1.5% agarose gel containing ethidium bromide using Tris-acetate EDTA buffer and photographed. A photograph of the agarose gel is shown in
WSSV Genome Organization
The genome of WSSV contains a circular double-stranded (ds) DNA of about 300 kb in size. So far three geographic isolates of WSSV have been completely sequenced including a Thai-isolate (292,967 bases, GenBank accession number AF369029, van Hulten et al., 2001, Virology 286:7-22), a Chinese isolate (305,307 bases, Yang et al., 2001, J. Virol. 75:11811-11820; GenBank accession number AF332093), and an isolate from Taiwan (307,287 bases, GenBank accession number AF440570). A representative genetic map of one of the isolates, Thai-isolate, is shown in
WSV is morphologically similar to insect baculovirus. However, phylogenetic analysis of ribonucleotide reductase and protein kinase genes revealed that WSV does not share a common ancestor with baculoviruses (van Hulten et al., 2000, J. Gen. Virol. 81:307-316; van Hulten et al., 2001b, Virus Genes 22:201-207). Due to its limited sequence similarity with other viruses sequenced so far, WSSV has been placed in a new virus family, Nimaviridae, genus Whispovirus, as described in the art, for example in the NCBI's publicly-available web site relating to viral taxonomy.
Transposable Elements in WSSV Genome
One of the three isolates of WSSV, the Taiwan isolate, contains a gene that encodes a transposase. The WSSV Taiwan isolate transposase gene shows 100% similarity with the homologous transposable elements of prokaryotic and eukaryotic origin. The regions upstream and the downstream of the ORF contains inverted repeats, a common feature of transposable elements. At the site of insertion of the transposable element in the WSSV genome, there has been a duplication of the WSSV sequence. The similarity of WSSV transposase with the homologous sequence in the GenBank database, and the genetic map of the transposase gene in WSSV genome are shown in
Identification of a Drosophila Homologue of Penelope Transposable Element in Shrimp
Transposable elements (TEs) have been identified from a wide variety of species including bacteria, plants, invertebrates, and vertebrates, and they constitute a considerable portion of the genetic makeup of a species. For example, TEs make up 3% of yeast genome (Kim et al., 1998), 10-15% of Drosophila sp. genome (SanMiguel et al., 1996, Science 274:765-768), and about 50% of maize genome (Pimpinelli et al., 1995). In human, TEs constitute 43% of the genome representing 4.3 million TEs (Smit, 1999, Curr. Opin. Genet. Dev. 9:657-663; Li et al., 2001, Nature 409:847-849). Although TEs have long been held as “selfish DNA”, only recently the importance of TEs is being realized (Kumar and Hirochika, 2001; Plasterk et al, 1999). TEs have been used in linkage mapping, genetic fingerprinting, molecular breeding, transgenic research, and evolutionary studies (Kumar et al., 2001, Trend Plant Biol. 6:127-134; Plasterk et al., 1999, Trends Genet.15:326-332).
Dhar and colleagues have recently reported the construction of a size fractionated genomic DNA library from shrimp (Penaeus monodon) for isolating microsatellite sequences for genetic analysis (Xu et al., 1999, Animal Genet. 30:1-7; Xu et al., 2004, J. Biochem. Genet., In press). GenBank database search for one of the DNA clones (GenBank accession number AF077579) showed significant similarity to a Drosophila virilis non-LTR transposable element Penelope (
Construction of a Shrimp Transient Expression Vector
In order to construct a shrimp transient expression vector, IHHNV P61 promoter was amplified from IHHNV-infected shrimp using genomic DNA as template for the PCR (
The entire TSV intergenic region between ORF1 and ORF2 is amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) following published protocol (Dhar et al., 2002). The cDNA amplicon is cloned into TOPO vector (Invitrogen, Inc, California) and recombinant clones are sequenced to confirm the identity of the cDNA. The primer sequences for amplifying the TSV intergenic region are: Forward 5′-TAGCA CCACC CGATC GTAAA C-3′ (SEQ ID NO: 21), Reverse 5′-TAATT AAGTC CCACC ACGCA AG-3′ (SEQ ID NO: 22).
After confirming the sequence, the insert for the TSV intergenic region is digested from the recombinant clone with appropriate restriction enzyme, run in a 1.5% agarose gel following standard protocol (Sambrook et al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor) and gel purified using the Qiagen gel purification kit (Qiagen, California).
The TSV intergenic region is cloned downstream of the IHHNV promoter. For this, the plasmid DNA from a recombinant clone containing the IHHNV promoter is digested with enzymes compatible with the TSV intergenic region insert and ligated using T4 DNA ligase and cloned in E. coli following standard protocol (Sambrook et al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor).
The recombinant clones containing the IHHNV P61 promoter and the TSV intergenic region are identified by colony PCR using IHHNVP61F primer and a vector derived primer. Clones that contain appropriate orientation (see
Assay of Shrimp Transient Expression In Vivo
Plasmid DNA from recombinant shrimp expression vector is injected into the tail muscle of juvenile shrimp (about 1 gram) and the activity of the reporter gene (DsRed) is assayed either directly using a transilluminator and direct observation of the shrimp or by excision of the area injected and then extraction of soluble protein and analysis on a fluorescent microplate reader (Matz et al., 1999, Nat. Biotechnol. 17:969-973). Shrimp injected with the plasmid DNA of a non-recombinant clone serve as a control. Injected shrimp are sacrificed at 24, 48, 72 hr post injection and the levels of DsRed protein is measured. A fluorescent microplate reader is used for this analysis.
Tissue is homogenized with a tissue homogenizer in 50 millimolar Tris (pH 7.0) buffer plus proteinase inhibitor (1 millimolar phenylmethylsulfonyl fluoride, 5 millimolar benzamidine). The soluble portion is recovered after a brief centrifugation. This is analyzed for total protein by the Lowry method and aliquotted into black Nunc microplates in serial dilution. Fluorescence is read on SpectraFluor Plus (Tecan) reader at 485 nm excitation and 535 emissions. Controls are run with standard DsRed protein either made internally or obtained commercially.
Assay of Shrimp Transient Expression In Vitro
Plasmid DNA from recombinant shrimp expression vector is injected to transfect primary cell culture of shrimp using lipofecting agent. Cells transfected with the plasmid DNA of non-recombinant clone serve as a control. The activity of the reporter gene (DsRed) is assayed at 24, 48, 72 hours post transfection following standard protocol (see Example 8).
Cloning of a Full-Length Shrimp Penelope Element
In order to clone a full-length shrimp Penelope element, RT-PCR is performed using DNase treated total RNA from Penaeus vannamei shrimp to amplify a 500 bp amplicon using shrimp Penelope primers. The primers are designed based on the partial sequence of shrimp Penelope element available in the GenBank database (accession number AF077579). The RT-PCR protocol for amplifying the Penelope element is same as described for amplifying other shrimp genes like beta-actin (Dhar et al., 2002). The amplified cDNA is run in a 1.5% agarose gel following standard protocol (Sambrook et al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor) and gel purified using Qiagen gel purification kit (Qiagen, California). The gel-purified cDNA is cloned in to a TOPO vector following manufacturer's recommendations (Invitrogen, Inc., California). Recombinant clones are sequenced to confirm the identity. The 500 bp partial clone of shrimp Penelope element is used as a probe to pull out a full-length clone of Penelope element from a shrimp (Penaeus vannamei) cDNA library (Dhar et al., 2004, “Comparison of expression profiles of healthy and white spot syndrome virus (WSSV) infected shrimp (Penaeus vannamei) by expressed sequence tag (EST) analysis,” Presented at the World Aquaculture Society meeting, Shrimp Genomics Section, March 1-5, 2004, Honolulu, Hi.). If the isolated clone lacks 5′-end, 5′-RACE technique is employed to clone the 5′-terminal sequences following protocol used to clone the 5′-terminal sequence of other shrimp gene (Roux et al., 2002, J. Virol. 76:7140-7149). Recombinant clones are sequenced to confirm the identity of the clone.
Construction of a Shrimp Transfection Vector Using a Shrimp Penelope Element
In order to construct a transfection vector, IHHNV P61 promoter (
The entire TSV intergenic region (TSV IRES) between ORF1 and ORF2 is amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) following published protocol (Dhar et al., 2002, J. Virol. Meth. 104:69-82). The cDNA amplicon is cloned into TOPO vector (Invitrogen, Inc, California) and recombinant clones are sequenced to confirm the identity of the cDNA. The primer sequences for amplifying the TSV intergenic region are: Forward: 5′-TAGCACCACC CGATCGTAAA C-3′ (SEQ ID NO: 23), Reverse 5′-TAATTAAGTC CCACCACGCA AG-3′ (SEQ ID NO: 24). After confirming the sequence, the insert for TSV IRES is digested from the recombinant clone with appropriate restriction enzyme, run in a 1.5% agarose gel following standard protocol (Sambrook et al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor) and gel purified using Qiagen gel purification kit (Qiagen, California). The TSV intergenic region is sub-cloned upstream of DsRed open reading frame in pDsRed vector (BD Bioscience, California). The recombinant clones containing the TSV intergenic region is sequenced to confirm the orientation and identity of the clones.
The TSV IRES-pDsRed plasmid is digested with two enzymes that cut upstream of the TSV IRES element. Similar enzymes are used to cut out the IHHNVP61-Penelope construct. The IHHNVP61-Penelope insert is gel purified using Qiagen gel purification kit before cloning upstream of the TSV intergenic region in pDsRed-TSV Intergenic construct. All enzyme digestion, ligation and transformation are done following standard protocol (Sambrook et al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor). Recombinant clones are sequenced to confirm the orientation and identity of the inserts.
Finally, the inverted terminal repeat elements of shrimp Penelope are amplified from shrimp full-length Penelope gene and cloned upstream of IHHNVP61 promoter and downstream of the selectable maker gene (in this case DsRed). A schematic representation of the final shrimp transfection vector is presented in
Construction of a Shrimp Transfection Vector Using a WSSV Transposable Element
In order to construct a transfection vector, IHHNV P61 promoter (
The entire TSV intergenic region (including the TSV IRES) between ORF1 and ORF2 is amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) following published protocol (Dhar et al., 2002, J. Virol. Meth. 104:69-82). The cDNA amplicon is cloned into a TOPO vector (Invitrogen, Inc, California) and recombinant clones are sequenced to confirm the identity of the cDNA. The primer sequences for amplifying the TSV intergenic region are: Forward: 5′-TAGCACCACC CGATCGTAAA C-3′ (SEQ ID NO: 25), Reverse 5′-TAATTAAGTC CCACCACGCA AG-3′ (SEQ ID NO: 26). After confirming the sequence, the insert for TSV IRES is digested from the recombinant clone with appropriate restriction enzyme, run in a 1.5% agarose gel following standard protocol (Sambrook et al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor) and gel purified using Qiagen gel purification kit (Qiagen, California). The TSV intergenic region is sub-cloned upstream of DsRed open reading frame in pDsRed vector (BD Bioscience, California). The recombinant clones containing the TSV intergenic region are sequenced to confirm the orientation and identity of the clones.
The TSV IRES-pDsRed plasmid is digested with two enzymes that cut upstream of the TSV IRES element. Similar enzymes are used to cut out the IHHNVP61-WSSV transposase construct. The IHNVP61-WSSV transposase is gel purified using Qiagen gel purification kit before cloning upstream of the TSV intergenic region in pDsRed-TSV Intergenic construct. All enzyme digestion, ligation and transformation are done following standard protocol (Sambrook et al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor). Recombinant clones are sequenced to confirm the orientation and identity of the inserts.
Finally, the inverted terminal repeat elements of WSSV are amplified by RT-PCR and cloned upstream of IHHNVP61 promoter and downstream of the selectable maker gene (in this case DsRed). A schematic representation of the final shrimp transfection vector is presented in
Transformation of shrimp primary cell culture with a transfection vector and assay of expression of recombinant protein in vitro
Plasmid DNA from a recombinant shrimp transfection vector is used to transfect primary cell culture of shrimp using a lipofecting agent. Shrimp primary cell culture for hemocytes is made following a published protocol (e.g., Itami et al., 1999, Meth. Cell Sci. 21:237-244). Shrimp hemocyte primary culture is transfected with the plasmid DNA of shrimp transfection vector described in Example 11,
In order to confirm the shrimp Penelope mediated integration of marker gene (DsRed), total genomic DNA is isolated from the shrimp transfected cells (primary cell lines) using Qiagen DNA extraction kit (Qiagen, California), digested with restriction enzymes, run in an agarose gel and blotted on to Nylon membrane (Sambrook et al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor). Southern hybridization is performed using TSV IRES probe and DsRed probe (Sambrook et al., 1989, “Molecular Cloning: A laboratory manual” Ed. 2, Cold Spring Harbor Press, Cold Spring Harbor). Positive hybridization signals using these probes indicate transformation of the shrimp DNA with the transfection vector construct.
Diagnostic Use of the Transgenic Cell Line of Example 13.
Adherent shrimp cells produced as in Example 13 are utilized for the diagnosis of viral disease by growth of the cells in a microplate. These cells are then exposed to shrimp tissue to be analyzed that has been macerated in a sterile tube and serially diluted using standard techniques. As infection proceeds, the fluorescence of the DsRed is monitored in a SpectraFluor Plus plate reader as described in Example 8. Cell mortality due to viral infection is reflected in a decrease in DsRed emission and viral titer can be determined rapidly versus a standard curve run simultaneously.
Construction of a IHHNV p61 and p2 Promoter Driven Luciferase Vectors.
The IHHNV p61 promoter element (GenBank accession # AF273215) was excised from a previously constructed and verified clone displayed in
Briefly, the targeted fragment was PCR amplified using the forward primer p61 1 F (5′-TAC AGA GCT CGG TAC CTC CAG CTG A-3′; SEQ ID NO: 27) and the reverse primer p61 1 R (5′-GCT AGC TAG CTT CGT ATT CTT GGA AGA GTC-3′; SEQ ID NO: 28). This amplicon was then digested with SacI and NheI restriction enzymes (underlined in primer sequences) for insertion. The digested amplicon was gel purified using methods described for MiniElute Gel Extraction Kit in the manufacturer's instructions (Qiagen, Valencia, Calif.). The purified fragment was then inserted into a pGL3-Basic reporter vector containing a modified coding region for firefly (Photinus pyralis) luciferase (Promega Corp. Madison, Wis.) that had been digested and gel purified as above (
Replicate clones were verified for sequence integrity at an off-site location using standard methods applied to an ABI 3100 capillary DNA analyzer (Applied Biosystems, Foster City, Calif.). The verified sequence was then excised out as before and inserted into the same location within a vector containing sequences reported to enhance expression of cloned fragments (pGL3-Enhancer, GenBank accession # U47297 (Cat# E1771, Promega Corp. Madison, Wis.). Again, replicate clones were verified as stated above. This produced the construct shown in
The p2 promoter element from IHHNV was synthesized using methods described previously (Smith et al., 2003, Proc. Natl. Acad. Sci. USA 100:15440-15445) at a commercial facility. Briefly, the synthesized fragment was inserted into the general use cloning vector pUC19 (GenBank accession # L09137). That plasmid was then sequence verified at the same location. Using the synthesized SacI and NheI sequences for excision, the p2 fragment was removed and inserted into the pGL3-Enhancer vector mentioned above. Replicate clones were verified as above. This produced the construct shown in
Construction of IHHNV p2 and p61 Promoter Driven DsRed2 Reporter Plasmid Construction
The IRES element from TSV (Cevallos et al., 2005, J. Virol. 79:677-683; GenBank accession # AF277675) was excised from a previously constructed and verified clone. Briefly, the targeted fragment was PCR amplified using the forward primer TSV IGR SACF (5′-GAT CGA GCT CTA GCA CCA CCC GAT CGT AAA C-3′; SEQ ID NO: 29) and TSV IGR BAMR (5′-GAT CGG ATC CTA ATT AAG TCC CAC CAC-3′; SEQ ID NO: 30). This amplicon was then digested with SacI and BamHI restriction enzymes (underlined in primer sequences) for insertion. The digested amplicon was gel purified using standard methods. The purified fragment was then inserted into a previously constructed and verified reporter vector pSTD (
An extended version of the TSV IGR was produced for cloning into the luciferase reporter vectors from above. The targeted fragment was PCR amplified using the forward primer TSV-IGR 2 F (5′-ATA CTC GAG AAC TAA TAG CAC CAC CCG-3′; SEQ ID NO: 31) and TSV-IGR 2 R (5′-TCC AAG CTT TTG TTG TAT CAA AAT TAT -3′; SEQ ID NO: 32), and electrophoresed in a 1.5% agarose gel (
Transient Expression of a Marker Protein in Shrimp.
Shrimp Handling.
Naive juvenile shrimp (Penaeus vannamei) free of specific pathogens were obtained from an outside source (Penaeus(Litopenaeus) vannamei (Kona line), about 1 gram, obtained from The Oceanic Institute. Waimanalo, Hi.). Specific Pathogen-Free (SPF) shrimp were free of those pathogens cited in Lightner (2003, “Exclusion of Specific Pathogens for Disease Prevention in a Penaeid Shrimp Biosecurity Program,” In “Biosecurity in Aquaculture Production Systems: Exclusion of Pathogens and Other Undesirables” The World Aquaculture Society, Baton Rouge, La.). The animals were held in artificial seawater at about 26 degrees Celsius containing standard sea salt components (INSTANT OCEANS® Cat# SS1-160P, Aquarium Systems Inc., Mentor, Ohio) until use. Shrimp Grower feed (2.4 millimeter 3/32, Cat# SI-35, Zeigler Bros., Inc. Gardners, Pa.) was given at regular intervals with the amount being about 3% of the total body weight. Excess feces and uneaten food were aspirated from the tanks when needed.
Shrimp were then distributed into 5 liter tanks (STERILITE® SHOW OFFS™, UPC#073149894366, Sterilite Corp. Townsend, Mass.) containing 4 liters of artificial seawater as above. Each group (two groups of 6 and four groups of 7) was handled according to the procedures above until experiment termination.
DNA Injection.
Using a 1 milliliter syringe, 50 microliters of solution (20% Glycerol, 0.9% NaCl, about 10 micrograms of purified plasmid DNA) was injected just under the carapace into the tail muscle of the animals. The injection site of each animal was the same. Animals were sacrificed after about 72 hours and screened for the presence of luciferase.
Tissue Lysate.
Animals were removed from the tanks and chilled in ice-cold water for about 5 minutes to anesthetize them. The tissue surrounding the injection site was excised and placed into a tube of standard lysis reagent supplemented with components for stabilization (1× Cell Culture Lysis Reagent; Cat# E153A, Promega Corp., Madison, Wis. 1× Protease Inhibitor Cocktail; Cat# P-2714, Sigma, St. Louis, Mo.). The excised tissue was homogenized with a pestle (1.5 milliliter microcentrifuge tube pestles; Cat# 01-1415-5390, USA/Scientific Plastics®, Milton Keynes, England) to release total cellular protein content into solution. The samples were then clarified through centrifugation and the supernatant removed and placed in a clean tube. The samples were held at 4 degrees Celsius until use.
Luciferase Assay.
All reactions listed here were performed in microplate format (96-well, white bottom; Cat# 655083, Greiner bio-one, Inc., Longwood, Fla.) using standardized reagents (BRIGHT-GLO™ luciferase assay substrate; Cat# E263A, Promega Corp., Madison, Wis.) and the luminescent signal quantified using a luminometer (SPECTRAFluor PLUS; Firmware-V 6.00 06-07-2003 Spectra; XFLUOR4 Version-V 4.50, Tecan US. Research Triangle Park, N.C.). The default settings for the luminometer were used. All reactions were carried out in a total volume of 100 microliters (50 microliters of sample +50 microliters of standardized reagent). Readings were made as soon as possible after the addition of reagent.
A standard curve using recombinant luciferase (QUANTILUM® recombinant luciferase. Source: North American firefly, Photinus pyralis; Cat# E170B, Promega Corp. Madison, Wis.) was constructed beginning at a concentration of 1 femtogram per microliter and serially diluted 2-fold to 15.6 attograms per microliter. Duplicate wells were averaged together for producing the graph in
Tissue lysate samples were read in triplicate wells. These technical replicate values were averaged together to form the chart in the data section below. Biological replicate values lying 1 standard deviation (SD) outside of the mean of the entire group were excluded. Standards were run to provide a plot of the standard curve values, shown in
Macerated tissue samples were treated as described above and assayed for luciferase expression. Shrimp luciferase data were extrapolated into the luciferase standard curve (Table 2). Luciferase activity was expressed as relative light units (RLU) for each biological replicate in the experiment and normalized to the amount of tissue excised.
The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.
While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention can be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include all such embodiments and equivalent variations.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/12979 | 4/15/2005 | WO | 6/5/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60562459 | Apr 2004 | US |
