Method of transposon-mediated mutagenesis in the nematode caenorhabditis elegans
The present invention provides methods for generating and identifying mutations in Caenorhabditis elegans . Disclosed herein are methods for introducing DNA into the C. elegans germline, methods for expressing DNA in the C. elegans germline, and methods for generating C. elegans mutants by using controlled mobilization of endogenous and heterologous transposons. Also disclosed are a transgene construct for expression in C. elegans and methods for inserting single copy DNA sequences into the C. elegans germline, and engineering mutations into the C. elegans genome.
1. A method of transposon-mediated mutagenesis in a C. elegans genome, comprising:
a. introducing a transgene construct into the C. elegans genome, wherein the construct comprises a transposase gene which is operably linked to a regulable expression control element and a 3′ untranslated region of a gene that is expressed in the C. elegans germline, wherein the 3′ untranslated region comprises a glh-2 3′ untranslated region; and
b. expressing the transposase gene, such that a transposon in the C. elegans genome transposes, causing a mutation in the C. elegans germline.
2. The method of claim 1 , wherein the transposon comprises a heterologous transposon.
3. The method of claim 2 , wherein the heterologous transposon is introduced into the C. elegans genome.
4. The method of claim 2 , wherein the transposon comprises a Mos 1 transposon.
5. The method of claim 2 , wherein the transposase gene comprises restriction sites 5′ of the start codon, restriction sites 5′ of the stop codon, and an artificial intron in the transposase gene open reading frame.
6. The method of claim 2 , wherein the regulable expression control element is an inducible promoter.
7. The method of claim 6 , wherein the promoter comprises a heat-shock promoter.
8. The method of claim 6 , wherein the promoter comprises a tetracycline-regulated promoter.
9. The method of claim 2 , wherein the construct is substantially free of bacterial plasmid DNA sequences.
10. The method of claim 2 , wherein the construct is substantially free of repeated DNA sequences.
11. The method of claim 2 , wherein the regulable expression control element comprises a glh-2 promoter.
12. The method of claim 1 , wherein the transposon comprises an endogenous transposon.
13. The method of claim 12 , wherein the transposon comprises a Tc3 transposon.
14. The method of claim 13 , wherein the transposase gene is a TC3A transposase gene.
15. The method of claim 12 , wherein the regulable expression control element is an inducible promoter.
16. The method of claim 15 , wherein the promoter comprises a heat-shock promoter.
17. The method of claim 15 , wherein the promoter comprises a tetracycline-regulated promoter.
18. The method of claim 1 , wherein the transposase gene is a TC3A transposase gene.
19. The method of claim 1 , wherein the construct is substantially free of bacterial plasmid DNA sequences.
20. The method of claim 1 , wherein the construct is substantially free of repeated DNA sequences.
21. The method of claim 1 , wherein the regulable expression control element comprises a heat-shock promoter.
22. The method of claim 1 , wherein the regulable expression control element comprises a glh-2 promoter.
23. The method of claim 1 , further comprising introduction of one or more additional copies of an endogenous transposon into the C. elegans germline.
24. The method of claim 23 , wherein the endogenous transposon is a Tc3 transposon.