Patent Application
20050260754
The present invention relates to constructs and methods for regulating the gene expression of at least two endogenous target genes by introducing, into a eukaryotic cell or a eukaryotic organism, an at least partially double-stranded ribonucleic acid molecule, the ribonucleic acid molecule comprising at least two ribonucleotide sequence segments which are homologous to various genes of the eukaryotic cell.
The specific inhibition of the gene expression of defined genes is one of those technologies of biotechnology which have been the subject of the most intense research. In this context, the expression of antisense RNA is the most widely used approach and described extensively (EP-A1 0 458 367; EP-A1 0 140 308; van der Krol A R et al. (1988) BioTechniques 6(10):658-676; de Lange P et al. (1995) Curr Top Microbiol Immunol 197:57-75, inter alia). However, antisense-RNA-mediated approaches have the disadvantage that stoichiometric amounts of the antisense RNA are required to bring about an effective inhibition of the target mRNA. Further problems are connected with the introduction, into the cells, of sufficient amounts of the antisense RNA and with the instability of the antisense. RNA. Antisense-RNA-based approaches are therefore inefficient in most cases.
A further approach for gene regulation is “co-suppression” meaning the reduction of the expression of an endogenous target gene by the recombinant expression of a sense RNA of this target gene (EP-A1 0 465 572). Co-suppression is believed to be based on more than one mechanism. The disadvantage is the lacking reliability and reproducibility of the method. In some cases, suppression takes place, while in other cases—caused by the expression of the sense RNA—the expected overexpression takes place. Also, the resulting phenotype is frequently not stable. The application of co-suppression is essentially limited to plants.
Various modifications of the methods based on antisense RNA or cosuppression are known. Thus, WO 93/23551 describes a method for inhibiting a plurality of genes by expressing a chimeric antisense RNA or sense RNA. The method cannot solve the usual problems connected with antisense RNA or sense RNA and remains inefficient.
WO 98/36083 and WO 99/15682 describe the regulation of gene expression by means of viral expression systems (“virus induced gene silencing” VIGS).
WO 99/32619 and WO 99/53050 describe methods for inhibiting individual target genes using an RNA with double-stranded structure, where the target gene and the region of the RNA duplex have at least partial identity with one another (see also: Montgomery MK et al. (1998) Proc Natl Acad Sci USA 95:15502-15507; Sharp P A (1999) Genes & Development 13(2):139-141; Fire A et al. (1998) Nature 391:806-11). The method is currently also referred to as “RNA interference” (RNAi) and its mechanism and action resembles the abovementioned VIGS method.
While the above-described methods, in particular the RNAi method, solve some of the problems in connection with the reduction of individual target genes, no satisfactory solution has been provided to date for other problems, in particular for the parallel suppression of a plurality of target genes. A large number of approaches in biotechnology require not only the reduction of an individual gene, but of a plurality of target genes, such as, for example, various genes of one or more metabolic pathways or whole gene families. To date, this could only be achieved with laborious and time-consuming approaches. The approaches frequently required the individual regulation of the individual target genes by successive transformation, for example using different expression constructs, each of which encoded an antisense RNA of a target gene. In addition to the fact that this is a considerably laborious and time-consuming approach, there is the disadvantage that only a limited number of selection markers, suitable promoters and the like is available for many systems and organisms, which makes multiple transformations considerably more difficult and requires for example the deletion of the markers after the transformation and selection. Frequently, the multiple use of a promoter has undesired consequences such as, for example, epigenetic gene silencing. Here, the multiple use of the control sequences leads to their inactivation, similar to the above-described cosuppression.
It is an object of the present invention to provide novel methods which make possible an efficient reduction of the expression, in a eukaryotic cell or a eukaryotic organism, of at least two endogenous target genes. We have found that this object is achieved by the present invention.
In a first aspect, the invention relates to a method for reducing the expression of at least two different endogenous target genes in a eukaryotic cell or a eukaryotic organism by introducing, into said eukaryotic cell or said eukaryotic organism, an at least partially double-stranded ribonucleic acid molecule, the double-stranded ribonucleic acid molecule comprising
A further aspect of the invention comprises an at least partially double-stranded ribonucleic acid molecule, where the double-stranded ribonucleic acid molecule comprises
A further aspect is the use of the double-stranded ribonucleic acid molecule according to the invention in one of the methods according to the invention.
The present invention overcomes the problems set out above and makes possible a rapid, particularly effective method for regulating the expression of various target genes. This results in particular in the following advantages:
Surprisingly, no troublesome interference between the individual ribonucleotide sequence segments was observed in the method according to the invention.
“Endogenous target gene of a eukaryotic cell or a eukaryotic organism” refers to any nucleic acid sequence in a eukaryotic cell, a eukaryotic organism or in a part, organ, tissue, seed and the like of same which is capable of transcription. This may take the form of naturally occurring or else artificially introduced sequences (such as, for example, transgenic sequences), with naturally occurring sequences being preferred. Naturally occurring sequences are preferred and comprise not only the homologous sequences of the eukaryotic cell or the eukaryotic organism, but also genes of pathogens which are present in the eukaryotic cell or the eukaryotic organism following infection by a pathogen. The target gene can be located in the chromosomal DNA or the DNA of the organelles (such as, for example, of the plastids, for example chloroplasts and the like) or else be located extrachromosomally in the cell. The naturally occurring, homologous sequences of the eukaryotic organism preferably comprise genes of same which are present in the genome in a stable manner, the term genome referring to the totality of the genetic information and comprising both the chromosomal and the plastid DNA. The endogenous target gene is preferably a gene which naturally occurs in the chromosomal DNA. Preferred genes are those whose reduced expression brings about a modified phenotype.
“Reduction of” or “to reduce” the expression of a target gene is to be understood in the broad sense in the present context and comprises the partial or essentially complete prevention or blocking of the expression of the target gene or the RNA, mRNA, rRNA, tRNA derived therefrom and/or of the protein product encoded by it in a cell or organism or a part, tissue, organ, cell or seed derived therefrom, which prevention or blockage is based on differing cell-biological mechanisms. A reduction for the purposes of the invention comprises the quantitative reduction of an RNA, mRNA, rRNA, tRNA expressed by the target gene and/or of the protein product encoded by it up to an essentially complete absence of these. In this context, the expression of a particular RNA, mRNA, rRNA, tRNA and/or of the protein product encoded by it, in a cell or an organism, is preferably reduced by more than 50%, especially preferably more than 80%, very especially preferably more than 90%, most preferably more than 95%, in comparison with the same cell or organism which has not been subjected to the method. In this context, the reduction can be determined by methods with which the skilled worker is familiar. Thus, the reduction of the protein quantity can be determined for example by an immunological detection of the protein. Moreover, biochemical techniques such as Northern hybridization, nuclease protection assay, reverse transcription (quantitative RT-PCR), ELISA (enzyme-linked immunosorbent assay), Western blotting, radioimmunoassay (RIA) or other immunoassays and fluorescence-activated cell analysis (FACS) can be employed. Depending on the type of the reduced protein product, its activity or the effect on the phenotype of the organism or the cell may also be determined.
“Protein quantity” refers to the amount of a particular polypeptide in an organism, a tissue, a cell or a cell compartment.
“Reduction” of the protein quantity refers to the quantitative reduction of the amount of a particular polypeptide in an organism, a tissue, a cell or a cell compartment—for example by the method according to the invention—in comparison with the wild type of the same genus and species to which this method has not been applied, under otherwise identical framework conditions (such as, for example, culture conditions, age, nutrient supply and the like). In this context, the reduction amounts to at least 10%, preferably at least 10% or at least 20%, especially preferably by at least 40% or 60%, very especially preferably by at least 70% or 80%, most preferably by at least 90% or 95%. Methods for determining the protein quantity are known to the skilled worker. Examples which may be mentioned are: the micro-Biuret method (Goa J (1953) Scand J Clin Lab Invest 5:218-222), the Folin-Ciocalteu method (Lowry O H et al. (1951) J Biol Chem 193:265-275) or measuring the absorption of CBB G-250 (Bradford M M (1976) Analyt Biochem 72:248-254).
“Different” in context with two endogenous target genes preferably means that the RNA or mRNA transcribed by the two endogenous target genes is not identical. Preferably, the homology of the RNA or mRNA transcribed by the two endogenous target genes is less than 90%, preferably less than 80%, especially preferably less than 70%, very especially preferably less than 60%, most preferably less than 50%, in each case over the entire length of the transcribed RNA or mRNA.
“At least partially double-stranded ribonucleic acid molecule” (hereinbelow dsRNA) means ribonucleic acid molecule which are entirely or partially double-stranded. Preferably, the ribonucleic acid sequence is predominantly completely double-stranded. “Predominantly completely double-stranded” means that at least 50%, preferably 70%, especially preferably 80%, very especially preferably 90%, of the bases in the molecule are present as a pair with another base of the dsRNA or can at least be theoretically present as a pair with another base, depending on the sequence of the dsRNA and the base pairing rules and, if appropriate, a prediction of the RNA secondary structure by means of a suitable computer algorithm.
“Essentially identical” means that a “sense” ribonucleotide sequence of the dsRNA may also have insertions, deletions and individual point mutations in comparison with the sequence of the “sense” RNA transcript of an endogenous target gene. Mutations comprise substitutions, additions, deletions, inversion or insertions of one or more bases of a nucleic acid sequence. Preferably, the homology between a “sense” ribonucleotide sequence of a dsRNA and at least part of the “sense” RNA transcript of an endogenous target gene amounts to at least 60%, preferably at least 70%, very especially preferably at least 90%, most preferably 95%. The sequences may also be identical with the corresponding sequence of the target gene. 100% sequence identity between the “sense” ribonucleotide sequence of the dsRNA and at least part of the “sense” strand of the transcript of an endogenous gene is preferred, albeit not necessarily required, for bringing about an efficient reduction of the expression of the endogenous gene. Individual mutations are tolerated. Accordingly, the method is tolerant to sequence deviations as may be present as the result of genetic mutations, polymorphisms or evolutionary divergences. Thus, for example, it is also possible to use a single dsRNA which has been generated starting from a particular endogenous gene, to suppress the expression of further homologous endogenous genes of the same organism or else the expression of homologous endogenous genes in other related species.
Homology is understood as meaning the extent of agreement between two nucleotide, ribonucleotide or protein sequences, which is preferably calculated by alignment with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al. (1997) Nucleic Acids Res. 25:3389ff), setting the following parameters:
The skilled worker realizes that thymine (T) in the DNA sequence is regarded as the equivalent of uracil (U) in the RNA sequence when calculating the homology between DNA (for example genes) and RNA.
“Part of the “sense” RNA transcript of an endogenous target gene” means fragments of an RNA or mRNA transcribed by an endogenous target gene. In this context, said part preferably has a sequence length of at least 10 bases, preferably at least 25 bases, especially preferably at least 50 bases, very especially preferably at least 100 bases, most preferably at least 200 bases or at least 300 bases. Also comprised is the complete transcribed RNA or mRNA.
As an alternative, an “essentially identical” dsRNA can also be defined as a nucleic acid sequence which is capable of hybridizing with a part of a transcript, preferably of the mRNA, of an endogenous target gene (for example in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA 50° C. or 70° C. for 12 to 16 hours or under different standard hybridization conditions).
“Standard hybridization conditions” is to be understood in the broad sense and refers to less stringent, but also—preferably—stringent hybridization conditions. Such hybridization conditions are described, inter alia, in Sambrook J, Fritsch E F, Maniatis T et al., in Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
For example, the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with approximately 2×SSC at 50° C.) and—preferably—those with high stringency (with approximately 0.2×SSC at 50° C., preferably at 65° C.) (20×SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0). Moreover, the temperature during the washing step can be raised from low-stringency conditions at room temperature, approximately 22° C., up to—preferably—higher-stringency conditions at approximately 65° C. Both parameters, salt concentration and temperature, can be varied simultaneously, or else one of the two parameters can be kept constant while only the other is being varied. During the hybridization, denaturing agents such as, for example, formamide or SDS, may also be employed. In the presence of 50% formamide, the hybridization is preferably carried out at 42° C. Some examples of conditions for hybridization and washing step are detailed hereinbelow:
“Essentially complementary” means that the “antisense” ribonucleotide sequences of the dsRNA may also have insertions, deletions and individual point mutations in comparison with the complement of the “sense” ribonucleotide sequences. Preferably, the homology is at least 80%, preferably at least 90%, very especially preferably at least 95%, most preferably 100%, between the “antisense” ribonucleotide sequences and the complement of the “sense” ribonucleotide sequences. Complement here means—in the manner with which the skilled worker is familiar—the counterstrand derived in accordance with the base pairing rules.
The double-stranded structure of the dsRNA can be formed starting from a single, fully or partially autocomplementary RNA strand (in which the abovementioned “sense” and “antisense” ribonucleotide sequences of the dsRNA are all linked covalently with one another) or starting from two RNA strands (in which the abovementioned “sense” and “antisense” ribonucleotide sequences of the dsRNA are located on separate strands) which are fully or partially complementary to one another. In the case of two separate strands, for example, all “sense” ribonucleotide sequences may be located on one strand, while all “antisense” ribonucleotide sequences are located on the other strand. However, the sequences can also be allocated in different ways to the two strands. The formation of the double-stranded structure can take place in-vitro, but also in-vivo, for example in the eukaryotic cell itself. Preferably, the dsRNA is present in the form of a single autocomplementary RNA strand.
The individual “sense” ribonucleotide sequences can form a double-stranded RNA structure with the corresponding, essentially complementary “antisense” ribonucleotide sequences by means of base pairing and form a subunit of the dsRNA.
In the case of an autocomplementary strand, there are various possibilities for the primary structures of the dsRNA. Those listed hereinbelow are to be understood as examples, but not by way of limitation:
If the dsRNA is—preferably—capable of forming a hairpin structure, the stem of the hairpin corresponds to the double-stranded portion of the dsRNA which is formed by base pairing between “sense” and “antisense” ribonucleotide sequence located on the same RNA molecule. Here, “sense” and “antisense” ribonucleotide sequences are preferably connected by a “linker”. The “linker” is preferably an intron, which can be spliced out of the dsRNA. Autocomplementary dsRNA structures starting from a single RNA molecule are preferred since they only require the expression of one construct and always comprise the complementary RNA strands in an equimolar ratio.
When using a linker (I)—preferably an intron—, the following schematic primary structures may be mentioned as examples of the dsRNA:
However, the dsRNA molecules are also functional without the linker. In this case, however, it must be taken into consideration that the last approx. 10 nucleotides of the terminal subunit S(n) no longer undergo correct pairing. In this case, the length for this subunit is to be complemented by 10 nucleotides. The linker is preferably an intron, especially preferably an intron in sense orientation. It is preferably an intron of a plant gene. The following may be mentioned by way of example, but not by limitation: the intron 3 of the maize alcohol dehydrogenase 1 (Adh1) (GenBank Acc.-NO.: AF044293; GI: 2828164), the intron 4 of the soya beta-conglycinin alpha subunit (GenBank Acc.-NO.: AB051865); one of the introns of the pea rbcS-3A gene for the ribulose-1,5-bisphosphate carboxylase (RBC) small subunit (GenBank Acc.-NO.: X04333). The skilled worker is familiar with these and further suitable introns (McCullough A J & Schuler M A (1997) Nuc Acids Res 25:1071-1077). For the application in the method according to the invention, the intron is preferably employed in combination with splice acceptor and splice donor sequences, which make it possible that the dsRNA is spliced out at a later point in time. These splice sequences can be the flanking sequences of the intron themselves, or else be provided by corresponding sequences of the remainder of the dsRNA.
Each of the individual “sense” ribonucleotide sequences of the dsRNA is essentially identical to at least part of the “sense” RNA transcript of an endogenous target gene. However, not all “sense” ribonucleotide sequences in this context are identical to the “sense” RNA transcript of an individual endogenous target gene, but the maximum identity in each case, of at least two of the “sense” ribonucleotide sequences, is with the “sense” RNA transcripts of different endogenous target genes. In this case, the homology between the transcripts of the two endogenous target genes is under 90%, preferably under 80%, especially preferably under 70%, very especially preferably under 60%, most preferably under 50%.
At least two of the individual “sense” ribonucleotide sequences comprised in the dsRNA according to the invention are different. Different means firstly that the target genes with whose transcripts they have in each case the maximum identity are not identical. Preferably, at least one subunit of the dsRNA reduces the expression of another gene as at least one other subunit. Secondly, different can also mean that the “sense” ribonucleotide sequences of the subunits themselves are essentially not identical and preferably have under 60%, more preferably under 50%, even more preferably under 40% homology with one another. In a further embodiment, the dsRNA can comprise more than one copy of a subunit. Moreover, the dsRNA can also comprise a plurality of different subunits which, however, are directed against the same endogenous target gene and whose “sense” ribonucleotide sequences are, for example, essentially identical to different parts of the “sense” RNA transcript of said endogenous target gene.
In this context, each of the individual “sense” ribonucleotide sequences can also be essentially identical to the transcript of a plurality of endogenous target genes. This is the case in particular when the target genes have similar sequence segments, as is the case, for example, in members of gene families (for example storage proteins). This is an especially advantageous use form since—when a suitable ribonucleotide sequence of a subunit is chosen—said subunit can reduce the expression of more than one target gene.
Preferably, the sequence of the dsRNA is chosen in such a way that the desired dsRNA structure has the in each case least free energy after formation of the duplex in comparison with other possible folding variants of the primary structure of the dsRNA. This can be ensured for example by avoiding sequence duplications and the like. The specific secondary structure can be predicted and optimized for example using suitable computer programs (for example FOLDRNA; Zuker and Stiegler (1981) Nucleic Acids Res 9(1):133-48).
In a preferred embodiment, each subunit of the dsRNA has a length of at least 20 base pairs, preferably at least 50 base pairs, especially preferably at least 100 base pairs, very especially referably at least 250 base pairs.
In a furthermore preferred embodiment, each unit has a length of an integer multiple of 21 or 22 base pairs, that is to say, for example, 21, 22, 42, 43, 44, 63, 64, 65, 66, 84, 85, 86, 87, 88, 105, 106, 107, 108, 109, 110, 126, 127, 128, 129, 131, 132, 147, 148, 149, 150, 151, 152, 153, 154, 168, 169, 170, 171, 172, 173, 174, 175, 176, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 or 220 base pairs, preferably 21, 22, 42, 44, 63, 66, 84, 88, 105, 110, 126, 132, 147, 154, 168, 176, 189, 198, 210 or 220 base pairs, very especially preferably 21, 42, 63, 84, 105, 126, 147, 168, 189 or 210 base pairs, most preferably 180 or 210 base pairs.
The “sense” and/or “antisense” ribonucleotide sequences of the individual subunits can be linked with one another directly or else linked with one another and/or flanked by a spacer (SP). Individual spacers (SP) can be identical or else different. The spacer preferably meets the same requirements with regard to length as have been detailed above for the length of the subunits themselves. The spacer can form a double-stranded structure, but may also exist—for example in the form of a bubble—in unpaired formation, i.e. the bases in strand and counterstrand need not necessarily be complementary. Preferred embodiments are described for example by the following primary structures:
The spacer can comprise further functional elements. The following may be mentioned by way of example, but not by limitation:
The dsRNA or its precursor molecules can be introduced into an organism or a cell in various ways with which the skilled worker is familiar. “To introduce” is to be understood in the broad sense and comprises, for the purposes of the present invention, all those methods which are suitable for directly or indirectly introducing, into an organism or a cell, compartment, tissue, organ or seed of same, a dsRNA or its precursor molecules, or generating it/them therein. The introduction can bring about the transient presence of a dsRNA, or else a stable presence. Comprised are methods of the direct transfection or transformation of the cell with the as well as the transformation or transfection of the cell with expression cassettes which are capable of expressing, in the cell, the ribonucleic acid sequences on which the dsRNA is based (hereinbelow dsRNA expression system). The expression of the dsRNA can be transient or—for example after integration into the genome of the organism—stable. The duplex formation of the dsRNA can be initiated either outside or within the cell.
The dsRNA is introduced in an amount which makes possible the presence of at least one copy per cell. Higher amounts (for example at least 5, 10, 100, 500 or 1000 copies per cell) can, if appropriate, effect a more efficient reduction of the expression of the target genes. Since dsRNA is extraordinarily mobile within an organism, it is not necessarily required to apply the dsRNA into each cell of the organism. It suffices to introduce, or to express, the dsRNA into a or few cells, it then being possible for the activity according to the invention also to be achieved in other cells of the same organism.
A dsRNA—for example for use in a direct transformation or transfection—can be synthesized in vivo or in vitro by enzymatic, molecular-biological or chemico-synthetic methods. Eukaryotic, prokaryotic or bacteriophage RNA polymerases (such as, for example, T3, T7 or SP6 RNA polymerase) can be used for this purpose. Suitable methods for the in-vitro expression of RNA are described (WO 97/32016; U.S. Pat. No. 5,593,874; U.S. Pat. No. 5,698,425, U.S. Pat. No. 5,712,135, U.S. Pat. No. 5,789,214, U.S. Pat. No. 5,804,693). Prior to introduction into a cell, tissue or organism, a dsRNA which has been synthesized in vitro, either chemically or enzymatically, can be purified either completely or in part from the reaction mixture, for example by extraction, precipitation, electrophoresis, chromatography or combinations of these methods. The dsRNA can be introduced directly into the cell (for example by particle bombardment or microinjection) or else be applied extracellularly (for example into the interstitial space, the vascular system, the digestion system and the like). An application of, for example, dsRNA-expressing organisms in the form of food is also feasible. It is known that dsRNA has good cell penetration characteristics and sufficient stability. Owing to the high efficacy of the dsRNA, only few molecules suffice to obtain a good effect for the purposes of the invention.
Furthermore, modifications of both the sugar-phosphate backbone and of the nucleosides may be present in the dsRNA. For example, the phosphodiester bonds of the RNA can be modified in such a way that they comprise at least one nitrogen or sulfur hetero atom. Bases can be modified in such a way that the activity, for example of adenosine deaminase is restricted. The dsRNA can be generated enzymatically or, fully or in part, chemico-synthetically.
However, the dsRNA is preferably expressed in the cell starting from suitable expression systems. A further aspect of the invention relates to said dsRNA expression systems. If the dsRNA is expressed as a single, autocomplementary RNA strand, the expression system comprises an expression cassette with a DNA sequence encoding the autocbmplementary RNA strand and in operable linkage with a promoter which is suitable for ensuring the expression in the eukaryotic cell in question. The expression cassette can optionally comprise further functional elements such as, for example, transcription terminators and/or polyadenylation signals. Such expression cassettes are likewise an aspect of the invention.
If the dsRNA is expressed in the form of two separate strands which are fully or partially complementary to one another, the expression system comprises two expression cassettes where each of the two strands is linked operably with a promoter which is suitable for ensuring the expression in the eukaryotic cell in question. The expression cassettes can optionally comprise further functional elements such as, for example, transcription terminators and/or polyadenylation signals. The two expression cassettes can be combined to give the expression system according to the invention in various ways as known to the skilled worker. Examples which may be mentioned are:
It is also possible to employ an expression cassette in which the dsRNA-encoding DNA sequence is located between two promoters with opposite direction of transcription, thus being transcribed from both sides.
Expression cassette refers to chimeric DNA molecules in which a nucleic acid sequence which encodes the dsRNA molecule (or one of the strands thereof) is linked to at least one genetic control element (for example a promoter, enhancer, silencer, splice donor, splice acceptor or polyadenylation signal) such that the transcription of the dsRNA molecule (or one of the strands thereof) in the eukaryotic cell or organism is ensured. Suitable advantageous constructions are described hereinbelow. Polyadenylation is possible, but not necessary, nor do elements for initiating a translation have to be present.
If the expression construct is to be introduced into a plant and the dsRNA is to be generated in plantae, plant-specific genetic control elements (for example plant-specific promoters) are preferred. However, the dsRNA can also be generated in other organisms or in vitro and then be introduced into the plant.
Operable linkage is understood as meaning, for example, the sequential arrangement of a promoter with the nucleic acid sequence to be transcribed and, if appropriate, further regulatory elements such as, for example, a terminator and/or polyadenylation signals in such a way that each of the regulatory elements can fulfill its function when the nucleic acid sequence is transcribed, depending on the arrangement of the nucleic acid sequences. To this end, a direct linkage in the chemical sense is not necessarily required. Genetic control sequences such as, for example, enhancer sequences, can also exert their function on the target sequence in positions which are further away, or indeed from other DNA molecules. Arrangements are preferred in which the nucleic acid sequence to be transcribed is positioned behind the sequence acting as promoter, so that both sequences are covalently linked to one another. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs. In a preferred embodiment, the nucleic acid sequence to be transcribed is located behind the promoter in such a way that the transcription start is identical with the desired beginning of the dsRNA.
An operable linkage and an expression cassette can be realized by means of customary recombination and cloning techniques as are described, for example, in Maniatis T, Fritsch E F and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Silhavy T J, Berman M L and Enquist L W (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Ausubel F M et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience and in Gelvin et al. (1990) In: Plant Molecular Biology Manual.
However, an expression cassette is also understood as meaning those constructions in which for example a nucleic acid sequence encoding a dsRNA is placed in such a way behind an endogenous promoter that the same effect occurs. Both approaches give expression cassettes for the purposes of the invention.
Promoters which can be used for the method according to the invention are, in principle, all natural promoters together with their regulation sequences, such as those mentioned above, as long as they ensure expression in the target organism. Moreover, synthetic promoters can also be used advantageously.
Further promoters which make possible the expression in further eukaryotes or in prokaryotes, such as, for example, E. coli bacteria, may be linked operably with the nucleic acid sequence to be expressed.
The nucleic acid sequences which are present in the expression cassettes or vectors according to the invention can be linked operably with further genetic control sequences, in addition to a promoter. The term genetic control sequence is to be understood in the broad sense and refers to all those sequences which have an effect on the materialization or the function of the expression cassette according to the invention. Genetic control sequences modify for example transcription in prokaryotic or eukaryotic organisms. Preferably, the expression cassettes according to the invention comprise a plant-specific promoter 5′-upstream of the respective nucleic acid sequence to be expressed recombinantly, and 3′-downstream a terminator sequence as additional genetic control sequence, and, if appropriate, further customary regulatory elements, in each case linked operably with the nucleic acid sequence to be expressed recombinantly.
Genetic control sequences furthermore also comprise the 5′-untranslated regions, introns or noncoding 3′-region of genes. It has been demonstrated that these may play an important role in the regulation of gene expression. Control sequences furthermore comprise polyadenylation signals and terminator sequences.
The expression cassette can advantageously comprise one or more of what are known as enhancer sequences in operable linkage with the promoter, which sequences make possible an increased recombinant expression of the nucleic acid sequence. Additional advantageous sequences such as further regulatory elements or terminators may also be inserted at the 3′-end of the nucleic acid sequence to be expressed recombinantly. One or more copies of the nucleic acid sequences to be expressed recombinantly may be present in the gene construct.
Control sequences are furthermore understood as being those sequences which make possible a homologous recombination or insertion into the genome of a host organism, or which permit the removal from the genome. Methods such as the cre/lox technology permit a tissue-specific, if appropriate inducible, removal of the expression cassette from the genome of the host organism (Sauer B (1998) Methods. 14(4):381-92). In this method, specific flanking sequences (lox sequences), which later allow removal by means of cre recombinase, are attached to the target gene.
Preferably, the expression cassette consisting of a linkage of promoter and nucleic acid sequence to be transcribed can be present integrated in a vector and can be introduced into the eukaryotic cell or organism by, for example, transformation, by one of the methods described hereinbelow. The subsequent expression can be transient or else—preferably—stable after insertion (for example using selection markers) of the expression cassettes into the genome. Preferably, the dsRNA expression system is integrated stably into the genome—for example the chromosomal DNA or the DNA of the organelles (for example the plastids, mitochondria and the like)—of a cell.
The introduction, into an organism or cells, tissues, organs, parts or seeds of same (preferably into plants or plant cells, tissues, organs, parts or seeds), of a transgenic expression cassette according to the invention can advantageously be carried out using vectors in which the transgenic expression cassettes are present. Examples of vectors can be plasmids, cosmids, phages, viruses or else agrobacteria. The transgenic expression cassettes can be inserted into the vector (preferably a plasmid vector) via a suitable restriction cleavage site. The resulting vector is first introduced into E. coli. Correctly transformed E. coli are selected, grown, and the recombinant vector is obtained by methods with which the skilled worker is familiar. Restriction analysis and sequencing can be employed for verifying the cloning step. Preferred vectors are those which make possible a stable integration of the expression cassette into the host genome.
The generation of a transformed organism (or of a transformed cell or tissue) requires introducing the relevant DNA (for example the expression vector) or RNA into the relevant host cell. A multiplicity of methods (Keown et al. (1990) Methods in Enzymology 185:527-537) is available for this method which is referred to as transformation (or transduction or transfection). Thus, for example, the DNA or RNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles. Also, the cell can be permeabilized chemically, for example using polyethylene glycol, so that the DNA can enter the cell by diffusion. The DNA can also be effected by protoplast fusion with other DNA-comprising units such as minicells, cells, lysosomes or liposomes. Electroporation is a further suitable method for introducing DNA, where the cells are permeabilized reversibly by an electrical impulse. Suitable methods are described (for example in Bilang et al. (1991) Gene 100:247-250; Scheid et al. (1991) Mol Gen Genet 228:104-112; Guerche et al. (1987) Plant Science 52:111-116; Neuhause et al. (1987) Theor Appl Genet 75:30-36; Klein et al. (1987) Nature 327:70-73; Howell et al. (1980) Science 208:1265; Horsch et al. (1985) Science 227:1229-1231; DeBlock et al. (1989) Plant Physiology 91:694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press Inc. (1989)).
A further aspect of the invention relates to cells which comprise one of the dsRNA molecules, expression systems, expression cassettes or expression vectors according to the invention. The cell may be derived from an organism or be present in same, but also refers to single-celled organisms such as microorganisms. The cell can be prokaryotic or of eukaryotic nature. The method according to the invention is applied to eukaryotic organisms. However, prokaryotic organisms may still comprise the expression systems according to the invention, for example for the purposes of dsRNA production. Also, prokaryotic organisms, for example agrobacteria, can advantageously be employed as vehicles for the transformation of, for example, plant organisms.
Preferred prokaryotes are mainly bacteria such as bacteria of the genus Escherichia, Corynebacterium, Bacillus, Clostrridium, Proionibacterium, Butyrivibrio, Eubacterium, Lactobacillus, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Phaeodactylum, Colpidium, Mortierella, Entomophthora, Mucor, Crypthecodinium or Cyanobacteria, for example of the genus Synechocystis. Microorganisms which are preferred are mainly those which are capable of infecting plants and thus of transferring the constructs according to the invention. Preferred microorganisms are those of the genus Agrobacterium and in particular the species Agrobacterium tumefaciens.
Eukaryotic cells and organisms comprises plant and animal, nonhuman organisms and/or cells and eukaryotic microorganisms such as, for example, yeasts, algae or fungi. A corresponding transgenic organism can be generated for example by introducing the expression systems in question into a zygote, stem cell, protoplast or another suitable cell which is derived from the organism.
“Animal organism” refers to nonhuman vertebrates or invertebrates. Preferred vertebrates comprise, for example, fish species, nonhuman mammals such as cattle, horse, sheep, goat, mouse, rat or pig, and birds such as chicken or goose. Preferred animal cells comprise CHO, COS, HEK293 cells. Invertebrates comprise nematodes or other worms, and insects. Invertebrates comprise insect cells such as Drosophila S2 and Spodoptera Sf9 or Sf21 cells.
Furthermore preferred are nematodes which are capable of attacking animals or humans such as those of the genera Ancylostoma, Ascaridia, Ascaris, Bunostomum, Caenorhabditis, Capillaria, Chabertia, Cooperia, Dictyocaulus, Haemonchus, Heterakis, Nematodirus, Oesophagostomum, Ostertagia, oxyuris, Parascaris, Strongylus, Toxascaris, Trichuris, Trichostrongylus, Tfhchonema, Toxocara or Uncinaria. Furthermore preferred are those which are capable of attacking plant organisms such as, for example, Bursaphalenchus, Criconemella, Diiylenchus, Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus, Melodoigyne, Nacobbus, Paratylenchus, Pratylenchus, Radopholus, Rotelynchus, Tylenchus or Xiphinema. Preferred insects comprise those of the genera Coleoptera, Diptera, Lepidoptera and Homoptera.
Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria or further fungi described in Indian Chem Engr. Section B. Vol 37, No 1,2 (1995), page 15, Table 6. Especially preferred is the filamentous Hemiascomycet Ashbya gossypii.
Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia, especially preferred are Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178).
Preferred as transgenic organisms are mainly plant organisms. “Plant organism” comprises any organism which is capable of photosynthesis, and the cells, tissues, parts or propagation material (such as seeds or fruits) derived therefrom. Encompassed within the scope of the invention are all genera and species of higher and lower plants of the Plant Kingdom. Annual, perennial, monocotyledonous and dicotyledonous plants and gymnosperms are preferred. Encompassed are mature plant, seed, shoots and seedlings, and parts, propagation material (for example tubers, seeds or fruits) and cultures, for example cell cultures or callus cultures, derived therefrom. Mature plants refer to plants at any developmental stage beyond the seedling stage. Seedling refers to a young, immature plant at an early developmental stage.
For the purposes of the invention, “plant” means all genera and species of higher and lower plants of the Plant Kingdom. This term includes the mature plants, seeds, shoots and seedlings, and parts, propagation material, plants organs, tissues, protoplasts, callus and other cultures, for example cell cultures, derived therefrom, and any other type of group of plant cells which give functional or structural units. Mature plants refer to plants at any developmental stage beyond the seedling stage. Seedling refers to a young, immature plant at an early developmental stage. “Plant” comprises all annual and perennial, monocotyledonous and dicotyledonous plants and includes by way of example, but not by limitation, those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea and Populus.
Preferred are plants of the following plant families: Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanacea, Sterculiaceae, Tetragoniacea, Theaceae, Umbelliferae.
Preferred monocotyledonous plants are selected in particular from among the monocotyledonous crop plants such as, for example, the family of the Gramineae, such as alfalfa, rice, maize, wheat or other cereal species such as barley, millet and sorghum, rye, triticale or oats, and sugar cane, and also all grass species.
The invention is very especially preferably applied to dicotyledonous plant organisms. Preferred dicotyledonous plants are selected in particular from among the dicotyledonous crop plants such as, for example,
Also encompassed are ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or turf. Plants which may be mentioned by way of example but not by limitation are angiosperms, bryophytes such as, for example, Hepaticae (liverwort) and Musci (mosses); pteridophytes such as ferns, horsetail and clubmosses; gymnosperms such as conifers, cycades, ginkgo and Gnetalae, the families of Rosaceae such as rose, Ericaceae such as rhododendron and azalea, Euphorbiaceae such as poinsettias and croton, Caryophyllaceae such as pinks, Solanaceae such as petunias, Gesneriaceae such as African violet, Balsaminaceae such as touch-me-not, Orchidaceae such as orchids, Iridaceae such as gladioli, iris, freesia and crocus, Compositae such as marigold, Geraniaceae such as geranium, Liliaceae such as dracena, Moraceae such as ficus, Araceae such as cheeseplant and many others.
Furthermore, plant organisms for the purposes of the invention are further organisms capable of being photosynthetically active such as, for example, algae, cyanobacteria and mosses. Preferred algae are green algae such as, for example, algae from the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Synechocystis is particularly preferred.
Most preferred are
Depending on the host organism, the organisms used in the method are grown or cultured in a manner with which the skilled worker is familiar. As a rule, microorganisms are grown in a liquid medium comprising a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium, and, if appropriate, vitamins, at temperatures of between 0° C. and 100° C., preferably between 10° C. to 60° C., while passing in oxygen. The pH of the liquid medium can be kept at a constant value, that is to say regulated during the culturing period, or else not. The culture can be batchwise, semibatchwise or continuous. Nutrients can be provided at the beginning of the fermentation or fed in semicontinuously or continuously.
The subsequent application of the method according to the invention may be mentioned by way of example, but not by limitation:
I. Plant Biotechnology
The method according to the invention is preferably employed for the purposes of plant biotechnology for generating plants with advantageous properties. Thus, the suitability of the plants or their seeds as foodstuff or feeding stuff can be improved, for example via a modification of the compositions and/or the content of metabolites, in particular proteins, oils, vitamins and/or starch. Also, growth rate, yield or resistance to biotic or abiotic stress factors can be increased. The subsequent applications in the field of plant biotechnology are particularly advantageous. The possible target genes stated are to be understood by way of example, but not by limitation:
Further examples of advantageous genes are mentioned for example in Dunwell J M, Transgenic approaches to crop improvement, J Exp Bot. 2000;51 Spec No; pages 487-96.
Each of the abovementioned applications can be used as such on its own. Naturally, it is also possible to use more than one of the abovementioned approaches simultaneously. If, in this context, all approaches are used, the expression of at least two differing target genes as defined above is reduced. In this context, these target genes can originate from a single group of genes which is preferred for a use, or else be assigned to different use groups.
For using the methods according to the invention, the skilled worker has available well-known tools, such as expression vectors with promoters which are suitable for plants, and methods for the transformation and regeneration of plants. Plant-specific promoters means principally any promoter which is capable of governing the expression of genes, in particular foreign genes, in plants or plant parts, plant cells, plant tissues, plant cultures. In this context, the expression can be for example constitutive, inducible or development-specific. The following are preferred:
The expression cassettes may also comprise a chemically inducible promoter (review: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48:89-108) by means of which the expression of the exogenous gene in the plant can be controlled at a particular point in time. Such promoters such as, for example, the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22:361-366), salicylic-acid-inducible promoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP 0 388 186), a tetracyclin-inducible promoter (Gatz et al. (1992) Plant J 2:397-404), an abscisic-acid-inducible promoter (EP 0 335 528) or an ethanol- or cyclohexanone-inducible promoter (WO 93/21334) can likewise be used.
Especially preferred promoters are constitutive and seed-specific promoters.
Genetic control sequences also encompass further promoters, promoter elements or minimal promoters, all of which are capable of modifying the expression-governing characteristics. Thus, for example, genetic control sequences can bring about the tissue-specific expression additionally as a function of certain stress factors. Suitable elements have been described, for example, for water stress, abscisic acid (Lam E and Chua N H, J Biol Chem 1991; 266(26): 17131-17135) and heat stress (Schoffl F et al., Molecular & General Genetics 217(2-3):246-53, 1989).
Genetic control sequences furthermore also comprise the 5′-untranslated regions, introns or noncoding 3′ region of genes, such as, for example, the actin-1 intron, or the Adh1-S introns 1, 2 and 6 (general reference: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994)). It has been demonstrated that they can play a significant role in the regulation of gene expression. Thus, it has been demonstrated that 5′-untranslated sequences can enhance the transient expression of heterologous genes. An example which may be mentioned of such translation enhancers is the tobacco mosaic virus 5′ leader sequence (Gallie et al. (−1987) Nucl Acids Res 15:8693-8711) and the like. They can furthermore promote tissue specificity (Rouster J et al. (1998) Plant J 15:435-440).
Polyadenylation signals which are suitable as control sequences are plant polyadenylation signals, preferably those which correspond essentially to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular of gene 3 of the T-DNA (octopin synthase) of the Ti plasmid pTiACHS (Gielen et al. (1984) EMBO J. 3:83-5 ff) or functional equivalents thereof. Examples of especially suitable terminator sequences are the OCS (octopin synthase) terminator and the NOS (nopalin synthase) terminator.
An expression cassettes and the vectors derived therefrom may comprise further functional elements. The term functional element is to be understood in the broad sense and means all those elements which have an effect on the generation, multiplication or function of the expression cassettes, vectors or transgenic organisms according to the invention. The following may be mentioned by way of example, but not by limitation:
To select cells which have successfully undergone homologous recombination, or else transformed cells, it is usually required additionally to introduce a selectable marker which confers, to the cells which have successfully undergone recombination, a resistance to a biocide (for example a herbicide), a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic. The selection marker permits the transformed cells to be selected from untransformed cells (McCormick et al. (1986) Plant Cell Reports 5:81-84).
A variety of methods and vectors for introducing genes into the genome of plants and for the regeneration of plants from plant tissues or plant cells are known (Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Fla.), chapter 6/7, pp. 71-119 (1993); White FF (1993) Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and Wu R, Academic Press, 15-38; Jenes B et al. (1993) Techniques for Gene Transfer, in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, pp. 128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42:205-225; Halford N G, Shewry P R (2000) Br Med Bull 56(1):62-73). These include for example those mentioned above. In the case of plants, the described methods for the transformation and regeneration of plants from plant tissues or plant cells are utilized for transient or stable transformation. Suitable methods are mainly the transformation of protoplasts by polyethylene glycol induced DNA uptake, the liposome-mediated transformation (such as, for example, described in U.S. Pat. No. 4,536,475), biolistic methods using the gene gun (“particle bombardment” method; Fromm M E et al. (1990) Bio/Technology. 8(9):833-9; Gordon-Kamm et al. (1990) The Plant Cell 2:603), electroporation, the incubation of dry embryos in DNA-comprising solution, and microinjection. In the case of these “direct” transformation methods, the plasmid used need not meet any particular requirements. Simple plasmids, such as those of the pUC series, pBR322, M13 mp series, pACYC184 and the like can be used. If intact plants are to be regenerated from the transformed cells, an additional selectable marker gene must be located on the plasmid.
In addition to these “direct” transformation techniques, transformation can also be carried out by bacterial infection by eans of Agrobacterium (for example EP 0 116 718), viral infection by means of viral vectors (EP 0 067 553; U.S. Pat. No. 4,407,956; WO 95/34668; WO 93/03161) or by means of pollen (EP 0 270 356; WO 85/01856; U.S. Pat. No. 4,684,611).
The strains Agrobacterium tumefaciens or Agrobacterium rhizogenes, which are in most cases used for the transformation of Agrombacterium, also one by bacterial infection by means of, comprise a plasmid (Ti or Ri plasmid) which is transferred to the plant following infection with Agrobaterium. Part of this plasmid, referred to as T-DNA (transferred DNA), is integrated into the genome of the plant cell. Alternatively, Agrobacterium can also be used to transfer, to plants, binary vectors (mini Ti plasmids) and to integrate them into the plant genome. The Agrobacterium-mediated transformation is best suited to dicotyledonous diploid plant cells, while the direct transformation techniques are suitable for any cell type. Methods for the Agrobacterium-mediated transformation are described, for example, in Horsch RB et al. (1985) Science 225:1229f. If Agrobacteria are used, the expression cassette is to be integrated into specific plasmids, either into a shuttle, or intermediate, vector or into a binary vector. If a Ti or Ri plasmid is to be used for the transformation, at least the right border, but in most cases the right and the left border, of the Ti or Ri plasmid T-DNA is linked flanking region with the expression cassette to be introduced.
It is preferred to use binary vectors for the Agrobacterium tranformation. Binary vectors are capable of replicating both in E. coli and in Agrobacterium. As a rule, they comprise a selection marker gene and a linker or polylinker flanked by the right and left T-DNA border sequence. They can be transformed directly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163:181-187). The selection marker gene permits a selection of transformed Agrobacteria and is, for example, the nptII gene, which confers resistance to kanamycin. The Agrobacterium which acts as host organism in this case should already comprise a plasmid with the vir region. This region is required for transferring the T-DNA to the plant cell. An Agrobacterium transformed thus can be used for the transformation of plant cells. The use of T-DNA for the transformation of plant cells has been studied and described extensively (EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam, Chapter V; An et al. (1985) EMBO J. 4:277-287). A variety of binary vectors are known and, in some cases, commercially available, such as, for example, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA; Bevan et al. (1984) Nucl Acids Res 12:8711), pBinAR, pPZP200 or PPTV.
The Agrobacteria transformed with such a vector can then be used in the known manner for the transformation of plants, in particular crop plants such as, for example, oilseed rape, for example by bathing scarified leaves or leaf segments in an Agrobacterial solution and subsequently growing them in suitable media. The transformation of plants by Agrobacteria is described (white FF, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38; Jenes B et al. (1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press, pp. 128-143; Potrykus (1991) Annu Rev Plant-Physiol Plant Molec Biol 42:205-225). Transgenic plants can be regenerated in the known manner from the transformed cells of the scarified leaves or leaf segments, and these transgenic plants comprise the above-described expression systems according to the invention integrated into them.
Stably transformed cells, i.e. those which comprise the introduced DNA integrated into the DNA of the host cell can be selected from untransformed cells when a selectable marker is component of the introduced DNA. A marker can be for example any gene which is capable of conferring a resistance to antibiotics or herbicides (such as kanamycin, G418, bleomycin, hygromycin or phosphinothricin and the like); (see above). Transformed cells which express such a marker gene are capable of surviving in the presence of concentrations of a relevant antibiotic or herbicide which destroy an untransformed wild type. Example are mentioned above and comprise preferably the bar gene which confers resistance to the herbicide phosphinothricin (Rathore K S et al. (1993) Plant Mol Biol 21(5):871-884), the nptII gene, which confers resistance to kanamycin, the hpt gene, which confers resistance to hygromycin, or the EPSP gene, which confers resistance to the herbicide glyphosate. The selection marker permits the selection of transformed cells from untransformed cells (McCormick et al. (1986) Plant Cell Reports 5:81-84). The plants obtained can be grown and hybridized in the customary manner. Preferably, two or more generations should be cultured to ensure that the genomic integration is stable and hereditary.
As soon as a transformed plant cell has been generated, an intact plant can be obtained using methods with which the skilled worker is familiar. Here, the starting material is, for example, callus cultures. Shoot and root development in these as yet undifferentiated cell masses can be induced in the known manner. The resulting plantlets can be planted and grown. Suitable methods are described (Fennell et al. (1992) Plant Cell Rep. 11: 567-570; Stoeger et al (1995) Plant Cell Rep. 14:273-278; Jahne et al. (1994) Theor Appl Genet 89:525-533).
The expression efficacy of the recombinantly expressed nucleic acids can be determined for example in vitro by shoot-meristem propagation using one of the above-described selection methods. Moreover, changes in the nature and level of the expression of a target gene and the effect on the phontype of the plant can be tested in greenhouse experiments using test plants.
Vectors for expression in E. coli are preferably pQE70, pQE60 and pQE-9 (QIAGEN, Inc.); pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene Cloning Systems, Inc.); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia Biotech, Inc.).
Preferred vectors for expression in eukaryotes comprise pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (Stratagene Inc.); pSVK3, pBPV, pMSG and pSVL (Pharmacia Biotech, Inc.). Inducible vectors which may be mentioned are pTet-tTak, pTet-Splice, pcDNA4/TO, pcDNA4/TO/LacZ, pcDNA6/TR, pcDNA4/TO/Myc-His/LacZ, pcDNA4/TO/Myc-His A, pcDNA4/TO/Myc-His B, pcDNA4/TO/Myc-His C, pVgRXR (Invitrogen, Inc.) or the pMAM series (Clontech, Inc.; GenBank Accession No.: U02443). These vectors already provide the inducible regulatory control element, for example for a chemically inducible expression of a DSBI enzyme. The nucleic acid sequence encoding a DSBI enzyme can be inserted directly into these vectors.
Vectors for expression in yeast comprise for example pYES2, PYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, PHIL-D2, PHIL-S1, pPIC3SK, pPIC9K and PA0815 (Invitrogen, Inc.).
Advantageous control sequences are for example the Gram-positive promoters amy and SPO2 and the yeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
Cloning vectors and techniques for the genetic manipulation of ciliates and algae are known to the skilled worker (WO 98/01572; Falciatore et al. (1999) Marine Biotechnology 1(3):239-251; Dunahay et al. (1995) J Phycol 31:10004-1012).
Selection markers which can be used are, in principle, many of the selection systems which are also preferred for plants. Especially preferred are for mammalian cell the neomycin (G418) resistance, the hygromycin resistance, the zeocin resistance or the puromycin resistance. The ampicillin resistance, the kanamycin resistance or the tetracyclin resistant are especially preferred for prokaryotes.
In principle, for the transformation of animal cell or of yeast cells, similar methods as the “direct” tranformation of plant cells are to be applied. In particular, methods such as the calcium-phosphate- or liposome-mediated transformation or else electroporation are preferred.
A further aspect of the invention relates to the use of the transgenic organisms according to the invention and of the cells, cell cultures, parts—such as, for example, in the case of transgenic plant organisms roots, leaves and the like—derived from them and transgenic propagation material such as seeds or fruits for the production of foodstuffs or feeding stuffs, pharmaceuticals or fine chemicals, such as, for example, enzymes, vitamins, amino acids, sugars, fatty acids, natural or synthetic flavorings, aromas and colorants. Especially preferred is the production of triacylglycerides, lipids, oils, fatty acids, starches, tocopherols and tocotrienols and carotenoids. Genetically modified plants according to the invention which can be consumed by humans and animals can also be used as foodstuffs or feeding stuffs, for example directly or after undergoing a processing which is known per se.
Sequences
6. SEQ ID NO: 6
1.
Insert from vector pCR2.1-sRNAi4 (1) (cf. Example 2d) and pCR2.1-sRNAi8 (2) (cf. Example 2e) encoding an AtCru3-, AtCRB- and At2S3-expression-suppressing dsRNA.
In the two constructs, the “sense” ribonucleotide sequences and the complementary “antisense” ribonucleotide sequences (symbolized by the upside-down letters) for the individual target genes to be suppressed (AtCru3; AtCRB, At2S3) are arranged differently. Hatched regions (I1, I2 etc.) constitute intron sequences (linkers).
2.
S1, S2, . . . S(n): “sense” ribonucleotide sequence AS1, AS2, . . . AS(n): “antisense” ribonucleotide sequence I: intron sequence
The individual “sense” ribonucleotide sequences and “antisense” ribonucleotide sequences can be arranged in such a way that, first, all “sense” ribonucleotide sequences are arranged one next to the other, thus virtually forming a “sense” strand, whereupon then all “antisense” ribonucleotide sequences are linked with one another to give an “antisense” strand (A and C).
Alternatively, the individual “sense” ribonucleotide sequences and “antisense” ribonucleotide sequences can be arranged in such a way that pairs of in each case complementary “sense” ribonucleotide sequences and “antisense” ribonucleotide sequences are linked with one another (B and D).
“Sense” ribonucleotide sequences and “antisense” ribonucleotide sequences can be linked directly with one another (A and B) or else be separated from one another by further sequences, for example introns (I) (C and D).
3.
S1, S2, . . . S(n): “sense” ribonucleotide sequence
AS1, AS2, . . . AS(n): “antisense” ribonucleotide sequence
SP: “SPACER”
RE: Recognition sequence for ribozyme
R: Ribozyme
“Sense” ribonucleotide sequences and “antisense” ribonucleotide sequences can be separated from one another by further sequences (“SPACER”; SP) (A). The spacer can be for example a recognition sequence for a ribozyme. The corresponding ribozyme can be expressed separately (B) or else likewise be encoded by the spacer (C).
4.
5.
General Methods:
Unless otherwise specified, all chemicals are obtained from Fluka (Buchs), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Restriction enzymes, DNA-modifying enzymes and molecular biology kits were from Amersham-Pharmacia (Freiburg), Biometra (Göttingen), Roche (Mannheim), New England Biolabs (Schwalbach), Novagen (Madison, Wis., USA), Perkin-Elmer (Weiterstadt), Qiagen (Hilden), Stratagen (Amsterdam, Netherlands), Invitrogen (Karlsruhe) and Ambion (Cambridgeshire, United Kingdom). The reagents used were employed in accordance with the manufacturer's instructions.
The chemical synthesis of oligonucleotides can be carried out for example in the known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The cloning steps carried out for the purpose of the present invention such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, bacterial cultures, propagation of phages and sequence analysis of recombinant DNA, are carried out as described in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. Recombinant DNA molecules are sequenced using an ABI laser fluorescence DNA sequencer by the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA 74:5463-5467).
The plant Arabidopsis thaliana represents a member of the Higher Plants (spermatophytes). This plant is closely related to other plant species from the Cruciferea family such as, for example, Brassica napus, but also with other plant families of the dicots. Owing to the high degree of homology of its DNA sequences, or polypeptide sequences, Arabidopsis thaliana can be employed as model plant for other plant species.
a) Culture of Arabidopsis Plants
The plants are grown either on Murashige-Skoog medium supplemented with 0.5% sucrose (ogas et al. (1997) Science 277:91-94) or on compost (Focks & Benning (1998) Plant Physiol 118:91-101). To obtain uniform germination and flowering conditions, the seeds are first planted out or sprinkled on compost and then stratified for two days at 4° C. After flowering, the pods are labelled. Then, the pods are harvested according to the labels, at an age of 6 to 20 days after flowering.
b) Isolation of Total RNA and Poly-A+ RNA from Plants
RNA, or polyA+ RNA is isolated for the generation of suppression constructs. RNA was isolated from pods of Arabidopsis plants using the following protocol: pod material aged 6 to 20 days after flowering was harvested and shock-frozen in liquid nitrogen. Prior to further use, the material was stored at −80° C. 75 mg of the material was ground to a fine powder in a cooled mortar and admixed with 200 μl of the lysis buffer from the Ambion RNAqueos kit. The total RNA was then isolated following the manufacturer's instructions. The RNA was eluted with 50 μl of elution buffer (Ambion) and the concentration was determined by absorption of a 1:100 dilution in a photometer (Eppendorf) at 260 nm. 40 μg/ml RNA corresponds to an absorption of 1. The RNA solutions were brought to a concentration of 1 μg/μl using RNAse-free water. The concentrations were verified by agarose gel electrophoresis. To isolate polyA+ RNA, oligo(dT) cellulose from Amersham Pharmacia was used in accordance with the manufacturer's instructions. RNA or polyA+ RNA was stored at −70° C.
c) Construction of the cDNA Library
To construct the cDNA library from Arabidopsis pod RNA, the first-strand synthesis was obtained using reverse transcriptase from murine leukemia virus (Clontech) and oligo-d(T) primers, while the second-strand synthesis was achieved by incubation with DNA polymerase I, Klenow enzyme and cleavage with RNAse H at 12° C. (2 hours), 16° C. (1 hour) and 22° C. (1 hour). The reaction was stopped by incubation at 65° C. (10 minutes) and subsequently tranferred to ice. Double-stranded DNA molecules were made blunt-ended with T4 DNA polymerase (Roche, Mannheim) at 37° C. (30 minutes). The nucleotides were removed by phenol/chloroform extraction and by means of Sephadex G50 centrifugation columns. EcoRI/XhoI adapters (Pharmacia, Freiburg, Germany) were ligated onto the cDNA ends by means of T4 DNA ligase (Roche, 12° C., overnight), recut with XhoI and phosphorylated by incubation with polynucleotide kinase (Roche, 37° C., 30 minutes). This mixture was subjected to separation on a low-melting agarose gel. DNA molecules over 300 base pairs were eluted from the gel, phenol-extracted, concentrated on Elutip D columns (Schleicher & Schüll, Dassel, Germany), ligated onto vector arms and packaged into lambda-ZAPII phages or lambda-ZAP express phages, using the Gigapack Gold kit (Stratagene, Amsterdam, Netherlands), using the manufacturer's material and instructions.
d) Isolation of Genomic DNA from Plants Such as Arabidopsis thaliana or Brassica napus (CTAB Method)
To isolate genomic DNA from plants such as Arabidopsis thaliana or Brassica napus, approximately 0.25 g of leaf material of young plants in the vegetative stage are comminuted in liquid nitrogen in a pestle and mortar to give a fine powder. The pulverized plant material, together with 1 ml of CTAB I buffer (CTAB: Hexadecyltrimethylammonium bromide, also referred to as Cetyltrimethylammonium bromide; Sigma Cat. NO.: H6269) at a temperature of 65° C. and 20 μl of β-mercaptoethanol, is placed into a prewarmed second mortar and, after complete homogenization, the extract is transferred into a 2 ml Eppendorf vessel and incubated for 1 hour at 65° C. with regular careful mixing. After the mixture has cooled to room temperature, it is extracted in 1 ml of chloroform/octanol (24:1, extracted by shaking with 1M Tris/HCl, pH 8.0) by slowly inverting, and centrifuged for 5 minutes at. 8,500 rpm (7,500×g) and room temperature to achieve phase separation. Thereafter, the aqueous phase is reextracted with 1 ml of chloroform/octanol, centrifuged and mixed carefully by inverting with 1/10 volume of CTAB II buffer which has been prewarmed to 65° C. Thereafter, the mixture is admixed with 1 ml chloroform/octanol mixture (see above) by careful rocking and centrifuged for 5 minutes at 8,500 rpm (7,500×g) and room temperature to reseparate the phases. The aqueous lower phase is transferred into a fresh Eppendorf vessel and the upper organic phase is recentrifuged in a fresh Eppendorf vessel for 15 minutes at 8,500 rpm (7,500×g) and room temperature. The resulting aqueous phase is combined with the aqueous phase of the previous centrifugation step, and all of the mixture is admixed with precisely the same volume of pre-warmed CTAB III buffer. This is followed by incubation at 65° C. until the DNA precipitates in the form of floccules. This may take up to 1 hour or can be carried out overnight by incubation at 37° C. The sediment resulting from the subsequent centrifugation step (5 min, 2000 rpm (500×g), 4° C.) is admixed with 250 μl CTAB IV buffer which has been prewarmed to 65° C. and incubated at 65° C. for at least 30 minutes or until all of the sediment has dissolved. To precipitate the DNA, the solution is subsequently mixed with 2.5 volumes of ice-cold ethanol and incubated for 1 hour at −20° C. Alternatively, the mixture can be mixed with 0.6 volume of isopropanol and centrifuged immediately for 15 minutes at 8,500 rpm (7,500×g) and 4° C., without further incubation. The sedimented DNA is washed by inverting the Eppendorf vessel twice with in each case 1 ml of 80% strength ice-cold ethanol, recentrifuged after each washing step (5 min, 0.8,500 rpm (7,500×g), 4° C.) and subsequently dried in the air for approximately 15 minutes. The DNA is subsequently resuspended in 100 μl of TE with 100 μg/ml RNase and incubated for 30 minutes at room temperature. After a further incubation phase overnight at 4° C., the DNA solution is homogeneous and can be used for subsequent experiments.
Solutions for CTAB:
In each case the following is added freshly prior to use: 2% β-mercaptoethanol (20 μl for 1 ml of solution I).
Starting from the genomic Arabidopsis thaliana DNA or cDNA, the following fragments of storage protein sequences were amplified via PCR by means of the oligonucleotides listed. The following PCR protocol was employed:
PCR program: Initial denaturation for 2 minutes at 95° C., then 35 cycles with 45 seconds at 95° C., 45 seconds at 55° C. and 2 minutes at 72° C. Final extension: 5 minutes at 72° C.
a) Starting Vector pCR2.1-AtCRU3-RNAi
Starting from genomic Arabidopsis thaliana DNA, the following oligonucleotide primer pair is used to amplify an exon region with the complete subsequent intron including the splice acceptor sequence of the 12S storage protein AtCRU3, which sequence follows the intron (base pair 1947 to 2603 of the sequence with the GenBank Acc.No: U66916):
The PCR product is cloned into the pCR2.1-TOPO vector (Invitrogen) following the manufacturer's instructions, resulting in the vector pCR2.1-1, and the sequence is verified.
For the sequence encoding the antisense strand of the dsRNA, the following primer pair is used to amplify only the same exon as above (base pair 1947 to 2384) from Arabidopsis thaliana cDNA:
The PCR product is cloned into the pCR2.1-TOPO vector (Invitrogen) following the manufacturer's instructions, resulting in the vector pCR2.1-2, and the sequence is verified.
0.5 μg of vector pCR2.1-1 are incubated with restriction enzyme BamHI (New England Biolabs) for 2 hours following the manufacturer's instructions and then dephosphorylated for 15 minutes with alkaline phosphatase (New England Biolabs). The vector prepared thus (1 μl) is then ligated with the fragment obtained from vector pCR2.1-2. To this end, 0.5 μg of vector pCR2.1-2 is digested for 2 hours with BamHI (New England Biolabs) and the DNA fragments are separated by gel electrophoresis. The 489 bp segment which has been obtained in addition to the vector (3.9 kb) is excised from the gel and purified using the “Gel purification” kit (Qiagen), following the manufacturer's instructions, and eluted with 50 μl of elution buffer. 10 μl of the eluate are ligated with vector pCR2.1-1 (see above) overnight at 16° C. (T4 ligase, New England Biolabs). The ligation products are then transformed into TOP10 cells (Stratagene) following the manufacturer's instructions and a suitable selection takes place. Positive clones are verified with the primer pair ONP1 and ONP2, using PCR. The resulting vector is referred to as pCR2.1-AtCRU3-RNAi. The nucleic acid sequence encoding the dsRNA is described by SEQ ID NO: 105.
b) Starting Vector pCR2.1-AtCRB-RNAi
Using the subsequent oligonucleotide primer pair, an exon region of the 12S storage protein AtCRB (SEQ ID NO: 117 or 118; base pair 601 to 1874 of the sequence with the GenBank Acc. No.: M37248) is amplified from Arabidopsis thaliana cDNA:
The PCR product is cloned into the vector pCR2.1-TOPO (Invitrogen) following the manufacturer's instructions, resulting in the vector pCR2.1-3, and the sequence is verified.
For the region which acts as linker, an intron with the relevant splice acceptor and donor sequences of the flanking exons (base pair 1874 to 2117 of the sequence with the GenBank Acc. No.: M37248) is amplified from Arabidopsis thaliana genomic DNA using the following primer pair:
The PCR product is cloned into the vector pCR2.1-TOPO (Invitrogen) following the manufacturer's instructions, resulting in the vector pCR2.1-4, and the sequence is verified.
The construct for AtCRB is generated in a similar strategy as described for AtCRU3. Vector pCR2.1-3 is incubated for 2 hours with XhoI (New England Biolabs) and dephosphorylated (alkaline phosphatase, New England Biolabs). Likewise, vector pCR2.1-4 is incubated in the same manner with XhoI, and the gel fragments are separated by gel electrophoresis. The relevant fragments are purified and ligated in the manner described for AtCRU3, resulting, after bacterial transformation, into the vector pCR2.1-AtCRB exon/intron. This vector is incubated for 2 hours with XbaI (NEB), subsequently for 15 minutes with Klenow fragment (NEB), then for 2 hours with SalI and, finally, treated for 15 minutes with alkaline phosphatase (NEB). In parallel, the vector pCR2.1-3 is incubated with BamHI (NEB), then for 15 minutes with Klenow fragment and subsequently for 2 hours with XhoI (NEB). After gel electrophoresis, the exon fragment of AtCRB is isolated, purified and employed for the ligation. The two fragments were then ligated, resulting in the vector pCR2.1-AtCRB-RNAi. The resulting vector is referred to as pCR2.1-AtCRB-RNAi. The nucleic acid sequence which encodes the dsRNA is described by SEQ ID NO: 107.
c) Starting Vector pCR2.1-At2S3-RNAi
Using the following oligonucleotide primer pair, an exon region of the 2S storage protein At2S3 (SEQ ID NO: 3 or 4; base pair 212 to 706 of the sequence with the GenBank Acc. No.: M22035) is amplified:
The PCR product is cloned into the vector pCR2.1-TOPO (Invitrogen) following the manufacturer's instructions, resulting in the vector pCR2.1-5, and the sequence is verified. For the region which acts as linker, the same intron as specified in b), amplified with the primers OPN 7 and OPN 8, is employed. The construct for At2S3 is generated in a similar strategy as described for AtCRU3. Vector pCR2.1-5 is incubated for 2 hours with with XhoI (New England Biolabs) and dephosphorylated (alkaline phosphatase, New England Biolabs). Likewise, vector pCR2.1-3 is incubated in the same manner with XhoI, and the gel fragments are separated by gel electrophoresis. The relevant fragments are purified and ligated in the manner described for AtCRU3, resulting, after bacterial transformation, into the vector pCR2.1-At2S3 exon/intron. This vector is incubated for 2 hours with SalI (NEB), subsequently for 15 minutes with Klenow fragment (NEB) and, treated finally, for 15 minutes with alkaline phosphatase (NEB). In parallel, the vector pCR2.1-5 is incubated with BamHI (NEB) and then for 15 minutes with Klenow fragment. After gel electrophoresis, the exon fragment of At2S3 is isolated, purified and employed for the ligation. The two fragments are then ligated, resulting in the vector pCR2.1-At2S3-RNAi. The nucleic acid sequence which encodes the dsRNA is described by SEQ ID NO: 109.
d) Generation of Super-Supression Construct 1
The vectors pCR2.1-AtCRU3-RNAi and pCR2.1-4 (see above) are incubated for 2 hours at 37° C. with the restriction enzymes XhoI and SalI, the DNA fragments are separated by agarose gel electrophoresis, and both the vector and the PCR insert from pCR2.1-4 are excised and purified with the “Gel purification” kit from Qiagen following the manufacturer's instructions and eluted with 50 μl of elution buffer. 1 μl of the vector eluate and 8 μl of the eluate of the PCR insert from pCR2.1-4 are employed for the ligation, resulting in the construct pCR2.1-sRNAi1. This vector is incubated for 2 hours with the restriction enzyme XhoI and then for 15 minutes with Klenow fragment.
The vector pCR2.1-AtCRB-RNAi (see above) is incubated for 2 hours with the enzyme EcoRI and likewise treated for 15 minutes with Klenow fragment. Both incubation mixtures are separated by gel electrophoresis and either the vector (pCR2.1-sRNAi1) or the insert (from pCR2.1-AtCRB-RNAi) are excised from the agarose gel and the DNA fragments are purified as described above. 1 μl of the eluate of the vector and 8 μl of the eluate from the insert are employed for the ligation and incubated overnight at 4° C. The resulting construct is referred to as pCR2.1-sRNAi2. The resulting vector is incubated with the enzyme XbaI and subsequently with Klenow fragment. The vector pCR2.1-4 is incubated with the enzymes EcoRV and XbaI and subsequently with Klenow fragment. After gel electrophoresis and gel purification, the fragment from pCR2.1-4 is ligated with the vector pCR2.1-sRNAi2, resulting in the construct pCR2.1-sRNAi3. The resulting vector is then incubated for 2 hours with the enzyme ApaI and then for 15 minutes with Klenow fragment. As insert, the vector pCR2.1-At2S3-RNAi is incubated for 2 hours with the enzyme EcoRI and then for 15 minutes with Klenow fragment. After gel electrophoresis and gel purification, the eluates are ligated, resulting in the vector pCR2.1-sRNAi4. The sRNAi4 fragment (SEQ ID NO: 144; cf.
The vector employed pSUN-USP is a binary vector for the transformation of plants, based on pBinAR (Hbfgen and Willmitzer (1990) Plant Science 66: 221-230). A tissue-specific expression in seed can be achieved using the tissue-specific promoter USP promoter.
e) Generation of Super-Supression Construct 2
Starting from Arabidopsis thaliana cDNA, a fragment from the storage protein AtCRU3 (SEQ ID NO: 111, 112) is amplified with the following oligonucleotide primer pair under the PCR conditions stated in Example 2:
The resulting fragment is cloned into the vector pCR2.1-TOPO vector (Invitrogen) following the manufacturer's instructions, resulting in pCR2.1-6, and the sequences are verified.
Starting from Arabidopsis thaliana cDNA, a fragment from the storage protein At253 (SEQ ID NO: 3, 4) is amplified with the following oligonucleotide primer pair under the PCR conditions stated in Example 2:
The resulting fragment is cloned into the vector pCR2.1-TOPO vector (Invitrogen) following the manufacturer's instructions, resulting in pCR2.1-7, and the sequences are verified.
Starting from pCR2.1-3, pCR2.1-4 (see Example 2) and pCR2.1-6 and pCR2.1-7, the constructs are then ligated with one another as follows: the vector pCR2.1-3 is incubated for 2 hours with EcoRV and subsequently dephosphorylated for 15 minutes with alkaline phosphatase. The vector pCR2.1-6 is incubated for 2 hours with the enzymes StuI and EcoRV and the PCR insert is isolated via gel electrophoresis and gel purification. Vector pCR2.1-3 and insert from pCR2.1-6 are then ligated overnight at 4° C., resulting in the construct pCR2.1-sRNAi5. This vector is then incubated with EcoRV and dephosphorylated and ligated with the StuI/EcoRV-incubated and gel-purified fragment from pCR2.1-7, resulting in the construct pCR2.1-sRNAi6. This vector is then incubated with XhoI and dephosphorylated. The vector pCR2.1-4 is incubated with SalI and XhoI and the insert from pCR2.1-4 is ligated with the prepared vector pCR2.1-sRNAi6, resulting in the construct pCR2.1-sRNAi7. Starting from pCR2.1-sRNAi7, a PCR is carried out with the subsequent primer pair under the conditions stated in Example 2:
The resulting PCR product is incubated with the enzymes XhoI and SalI. The fragment is then ligated into the vector pCR2.1-sRNAi7 (incubated with XhoI), resulting in the construct pCR2.1-sRNAi8. The sRNAi8 fragment (SEQ ID NO: 146; cf.
The Agrobacterium-mediated transformation of plants can be carried out for example using the Agrobacterium tumefaciens strains GV3101 (pMP90) (Koncz and Schell (1986) Mol Gen Genet 204: 383-396) or LBA4404 (Clontech). The transformation can be carried out by standard transformation techniques (Deblaere et al. (1984) Nucl Acids Res 13:4777-4788).
The Agrobacterium-mediated transformation of plants can be carried out using standard transformation and regeneration techniques (Gelvin, Stanton B., Schilperoort, Robert A., Plant Molecular Biology Manual, 2nd ed., Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R., Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 pp., ISBN 0-8493-5164-2).
The transformation of Arabidopsis thaliana by means of Agrobacterium is carried out by the method of Bechthold et al., 1993 (C.R. Acad. Sci. Ser. III Sci. Vie., 316, 1194-1199). Oil-seed rape can be transformed for example by cotyledon or hypocotyl transformation (Moloney et al., Plant Cell Report 8 (1989) 238-242; De Block et al., Plant Physiol. 91 (1989) 694-701). The use of antibiotics for selection of Agrobacterium and plants depends on the binary vector used for the transformation and the Agrobacterium strain. The selection of oilseed rape is usually carried out using kanamycin as selectable plant marker.
Agrobacterium-mediated gene transfer into linseed (Linum usitatissimum) can be carried out using, for example, a technique described by Mlynarova et al. (1994); Plant Cell Report 13:282-285.
The transformation of soybean can be carried out using, for example, a technique described in EP-A-0 0424 047 (Pioneer Hi-Bred International) or in EP-A-0 0397 687, U.S. Pat. No. 5,376,543, U.S. Pat. No. 5,169,770 (University Toledo).
The transformation of plants using particle bombardment, polyethylene-glycol-mediated DNA uptake or via the silicon carbonate fiber technique is described for example by Freeling and Walbot “The maize handbook” (1993) ISBN 3-540-97826-7, Springer Verlag New York).
The activity of a recombinant gene product in the transformed host organism was measured at transcription and/or translation level.
A suitable method for determining the amount of transcription of the gene (which indicates the amount of RNA available for the translation of the gene product) is to carry out a Northern blot as detailed hereinbelow (by way of reference, see Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York, or the abovementioned example section), where a primer which is designed in such a way that it binds to the gene of interest is labeled with a detectable label (usually a radioactive label or chemiluminescent label) so that, when the total RNA of a culture of the organism is extracted, separated on a gel, transferred to a stable matrix and incubated with this probe, the binding and the extent of the binding of the probe indicate the presence and also the amount of the mRNA for this gene. This information also indicates the degree of transcription of the transformed gene. Cellular total RNA can be prepared from cells, tissues or organs in a plurality of methods, all of which are known in the art, such as, for example, the method described by Bormann, E. R., et al. (1992) Mol. Microbiol. 6:317-326.
Northern Hybridization:
To carry out the RNA hybridization, 20 μg of total RNA or 1 μg of poly(A)+ RNA are separated by means of gel electrophoresis in agarose gels with a strength of 1.25% using formaldehyde, as described in Amasino (1986, Anal. Biochem. 152, 304), capillary-blotted to positively charged nylon membranes (Hybond N+, Amersham, Braunschweig) using 10×SSC, immobilized by means of UV light and prehybridized for 3 hours at 68° C. using hybridization buffer (10% dextran sulfate w/v, 1 M NaCl, 1% SDS, 100 mg herring sperm DNA). The DNA probe was labeled with the Highprime DNA labeling kit (Roche, Mannheim, Germany) during the prehybridization, using alpha-32P-dCTP (Amersham Pharmacia, Braunschweig, Germany). After the labeled DNA probe had been added, the hybridization was carried out in the same buffer at 68° C. overnight. The washing steps were carried out twice for 15 minutes using 2×SSC. and twice for 30 minutes using 1×SSC, 1% SDS, at 68° C. The sealed filters were exposed at −70° C. over a period of 1 to 14 days.
Standard techniques, such as a Western blot, can be employed for assaying the presence or the relative amount of protein translated by this mRNA (see, for example, Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York). In this method, the cellular total proteins are extracted, separated by means of gel electrophoresis, transferred to a matrix such as nitrocellulose, and incubated with a probe, such as an antibody, which binds specifically to the desired protein. This probe is usually provided with a chemiluminescent or calorimetric label which can be detected readily. The presence and the amount of the label observed indicates the presence and the amount of the desired mutated protein which is present in the cell.
The effect of the genetic modification in plants, fungi, algae, ciliates, or on the production of a desired compound (such as a fatty acid) can be determined by growing the modified micro-organisms or the modified plants under suitable conditions (such as those described above) and analyzing the medium and/or cellular components for the increased production of the desired product (i.e. of lipids or of a fatty acid). These analytical techniques are known to the skilled worker and comprise spectroscopy, thin-layer chromatography, various types of staining methods, enzymatic and microbiological methods, and analytical chromatography such as high-performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) “Applications of HPLC in Biochemistry” in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3, chapter III: “Product recovery and purification”, pp. 469-714, VCH: Weinheim; Belter, P. A., et al. (1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992) Recovery processes for biological Materials, John Wiley and Sons; Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations, in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3; chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).
In addition to the abovementioned methods, plant lipids are extracted from plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96 (22):12935-12940, and Browse et al. (1986) Analytic Biochemistry 152:141-145. The qualitative and quantitative analysis of lipids or fatty acids is described in Christie, William W., Advances in Lipid Methodology, Ayr/Scotland: Oily Press (Oily Press Lipid Library; 2); Christie, William W., Gas Chromatography and Lipids. A Practical Guide—Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 pp. (Oily Press Lipid Library; 1); “Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952)-16 (1977) under the title: Progress in the Chemistry of Fats and Other Lipids CODEN.
In addition to measuring the end product of the fermentation, it is also possible to analyze other components of the metabolic pathways which are used for producing the desired compound, such as intermediates and by-products, in order to determine the overall efficiency of the production of the compound. The analytical methods encompass measurements of the nutrient quantities in the medium (for example sugars, carbohydrates, nitrogen sources, phosphate and other ions), measurements of the biomass composition and of the growth, analysis of the production of customary metabolites of biosynthetic pathways, and measurements of gases produced during fermentation. Standard methods for these measurements are described in Applied Microbial Physiology; A Practical Approach, P. M. Rhodes and P. F. Stanbury, ed., IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and references cited therein.
One example is the analysis of fatty acids (abbreviations: FAME, fatty acid methyl esters; GC-MS, gas-liquid chromatography/mass spectrometry; TAG, triacylglycerol; TLC, thin-layer chromatography).
Unambiguous proof for the presence of fatty acid products can be obtained by analyzing recombinant organisms by analytical standard methods: GC, GC-MS or TLC, as described variously by Christie and the references cited therein (1997, in: Advances on Lipid Methodology, fourth edition: Christie, Oily Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-verfahren [gas-chromatographic/mass-spectrometric methods], Lipide 33:343-353).
The material to be analyzed can be disrupted by sonication, milling in the glass mill, liquid nitrogen and milling or other applicable methods. After disruption, the material must be centrifuged. The sediment is resuspended in distilled water, heated for 10 minutes at 100° C., cooled on ice and recentrifuged, followed by extraction in 0.5 M sulfuric acid in methanol with 2% dimethoxypropane for 1 hour at 90° C., which gives hydrolyzed oil and lipid compounds, which give transmethylated lipids. These fatty acid methyl esters are extracted in petroleum ether and finally subjected to GC analysis using a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 micrometers, 0.32 mm) at a temperature gradient of between 170° C. and 240° C. for 20 minutes and for 5 minutes at 240° C. The identity of the fatty acid methyl esters obtained must be defined using standards which are available from commercial sources (i.e. Sigma).
The following protocol was used for the oil analysis of the Arabidopsis plants transformed with the suppression constructs: Lipid extraction from the seeds is carried out by the method of Bligh & Dyer (1959) Can J Biochem Physiol 37:911. To this end, 5 mg of Arabidopsis seeds are weighed into 1.2 ml Qiagen microtubes (Qiagen, Hilden) using a Sartorius (Gbttingen) microbalance. The seed material is homogenized with 500 μl chloroform/methanol (2:1; contains mono-C17-glycerol from Sigma as internal standard) in an MM300 Retsch mill from Retsch (Haan) and incubated for 20 minutes at RT. The phases were separated after addition of 500 μl 50 mM potassium phosphate buffer pH 7.5. 50 μl are removed from the organic phase, diluted with 1500 μl of chloroform, and 5 μl are applied to Chromarods SIII capillaries from Iatroscan (SKS, Bechenheim). After application of the samples, they are separated in a first step for 15 mins in a thin-layer chamber saturated with 6:2:2 chloroform:methanol: toluene. After the time has elapsed, the capillaries are dried for 4 minutes at room temperature and then placed for 22 minutes into a thin-layer chamber saturated with 7:3 n-hexane:diethyl ether. After a further drying step for 4 minutes at room temperature, the samples are analyzed in an Iatroscan MK-5 (SKS, Bechenheim) following the method of Fraser & Taggart, 1988 J. Chromatogr. 439:404. The following parameters were set for the measurements: slice width 50 msec, threshold 20 mV, noise 30, skim ratio 0. The data were quantified with reference to the internal standard mono-C17-glycerol (Sigma) and a calibration curve established with tri-C17-glycerol (Sigma), using the program ChromStar (SKS, Beichenheim).
To carry out the quantitative determination of the oil content, seeds of in each case 10 plants of the same independent transgenic line are analyzed. In total, the oil content of 30 transgenic lines of the T1 generation, 10 transgenic lines with in each case 10 plants of the T2 generation and 5 transgenic lines with in each case 10 plants of the T3 lines was determined. The transgenic plants showed a significantly higher oil content than corresponding control plants which had undergone the same treatment.
To assay the functionality of the multiple RNAi constructs, genes ere chosen whose suppression bring about a pronounced phenological effect. An example of such a gene is Toc159. This gene is essential for the development and functionality of chloroplasts in Arabidopsis (Bauer et al. Nature, 403, 203-207). Disruption of this gene leads to chlorophyll-deficient plants hose foliar phenotype is thus pale green to white. This albino phenotype can be distinguished very easily from normal plants.
GFP, the green fluorescent protein from the jellyfish Aequorea victoria was employed as further optical reporter gene. This reporter gene is a frequently used reporter gene in plants (see, for example, Stewart, Plant Cell Rep 2001 20(5):376-82). Arabidopsis thaliana cDNA or from the plasmid pEGFP (BD Clontech, Heidelberg, Genbank Accession U476561), was generated via PCR by means of the oligonucleotides mentioned. The following protocol was employed:
Composition of the PCR mix (50 μl):
PCR program: Initial denaturation for 2 minutes at 95° C., then 35 cycles of 45 seconds at 95° C., 45 seconds at 55° C. and 2 minutes at 72° C. Final extension of 5 minutes at 72° C.
a) Starting vector pGEM-Tocl59: Starting from Arabidopsis cDNA, the following oligonucleotide primer pair was used to amplify a fragment from Toc159 (Genbank Acc. No. T14P8.24):
The PCR product was cloned into the vector pGEM-T easy from Promega (Mannheim) following the manufacturer's instructions, resulting in the vector pGEM-Toc159, and the sequence was verified.
b) Starting vector pGEM-GFP: Starting from the plasmid pEGFP (BD lontech, Heidelberg, Genbank Acc.No.: U476561), the following oligonucleotide primer pair was used to amplify a fragment from GFP:
The PCR product was cloned into the vector pGEM-T easy from Promega (Mannheim) following the manufacturer's instructions, resulting in the vector PGEM-GFP, and the sequence was verified.
c) Generation of the construct pGEM-159-GFP. The vector PGEM-GFP was incubated for 2 hours with the restriction enzymes HindIII and BamHI. In parallel, the vector pGEM-Toc159 was incubated with the same restriction enzymes and subsequently then additionally treated for 15 minutes with alkaline phosphatase. The alkaline phosphatase was subsequently inactivated by heating at 95° C. for 10 minutes. The resulting DNA fragments from the two mixtures were separated via agarose gel electrophoresis. The 558 bp fragment from PGEM-GFP and the 3471 bp fragment from pGEM-Toc159 were excised from the gel and purified using the “gel purification” kit (Qiagen) following the manufacturer's instructions. The two fragments were ligated for 2 hours at 16° C. (T4 Ligase, New England Biolabs) and subsequently transformed into E. coli DH5a cells (Stratagen) following the manufacturer's instructions. Positive clones were“−”dentified by PCR using the primer pair OPN1 and OPN4 and subsequently verified by sequencing. The resulting vector was referred to as pGEM-159-GFP.
d) Generation of the supression construct 1: The vector pGEM-159-GFP was firstly incubated with the restriction enzymes XhoI and BamHI, and a further mixture was incubated with BamHI and SalI. The second mixture with BamHI/SalI was subsequently incubated for a further 15 minutes with alkaline phosphatase. The DNA fragments from the two mixtures were separated via agarose gel electrophoresis, and the following fragments were excised: mixture BamHI-XhoI, the 1091 bp fragment; mixture BamHI-SalI, the 4029 bp fragment. Both fragments were isolated from the agarose gel (see above) and then incubated for 2 hours at 16° C. with T4 ligase and subsequently transformed into E. coli DH5a cells (Stratagen). Positive clones were identified by PCR using the primer pair OPN1 and subsequently verified by sequencing. The resulting vector was termed suppression construct 1.
e) Generation of the suppression construct 2: The suppression construct 1 and the vector p3300.1 (Andreas Hilbrunner, PhD thesis ETH Zürich, 2003) were incubated for 2 hours with the restriction enzyme EcoRI. The vector p3300.1 was subsequently treated for 15 minutes with alkaline phosphatase. The two ixtures were mixed and incubated for 2 hours at 16° C. with T4 ligase. The ligation mix was then transformed into E. coli DH5α cells (Stratagen). The resulting suppression construct 2 was then employed for the transformation of Agrobacterium and of plants. The nucleic acid sequence encoding supression construct 2 (p3300.1-Toc159-GFP—RNAi) is shown in SEQ ID NO: 122.
The transformtion of agrobacteria and plants was carried out as described in Example 3 and 4, respectively. To assay the functionality of the suppression construct 2, the latter was transformed into Arabidopsis using the floral transformation method described by Bechtold et al., 1993 (C.R. Acad. Sci. Ser. III Sci. Vie., 316, 1194-1199). Arabidopsis plants of the variety Columbia-0 which already comprise the T-DNA of the bibary vector pBIN-35S-GFP were used as starting material.
In these plants, the green fluorescence of GFP is excited by excitation by ultraviolet light in the wavelength range 470-490 nm, thereby allowing the expression of the transgene which has been introduced. To this end, seedlings 1 week after germination or else leaf segments in older plants were analyzed using the Leica fluorescence microscope MZFLIII. The following parameters were set for the excitation of GFP: mercury lamp HBO 100W/DC, filter GFP3, image processing Leica software. The GFP analysis of green foliar material is made possible specifically by the use of a filter (GFP3) which does not allow transmission above a wavelength of 525 nm. Without this filter, it would not be possible to eliminate the pronounced autofluorescence of the leaf pigment chlorophyll. The Arabidopsis line used for the transformation revealed pronounced GFP expression after analysis under the microscope.
Transformed seeds were sown directly onto the ground and cultivated. After one week a check was made for sprouts having none, or a reduced proportion, of the leaf pigment chlorophyll. Such plants were easy to differetiate owing to their pale green or white phenotype.
These plants were then studied further under the fluorescence microscope and compared with corresponding green plants which had been grown in parallel. As an example,
Analysis of the albino plants under the fluorescence microscope then revealed (
| Number | Date | Country | Kind |
|---|---|---|---|
| 102 12 892.8 | Mar 2002 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP03/02735 | 3/17/2003 | WO | 9/20/2004 |
