Homologous gene targeting in Caenorhabditis elegans by biolistic transformation - PubMed (original) (raw)
Homologous gene targeting in Caenorhabditis elegans by biolistic transformation
Eugene Berezikov et al. Nucleic Acids Res. 2004.
Abstract
Targeted homologous recombination is a powerful approach for genome manipulation that is widely used for gene alteration and knockouts in mouse and yeast. In Caenorhabditis elegans, several methods of target-selected mutagenesis have been implemented but none of them provides the opportunity of introducing exact predefined changes into the genome. Although anecdotal cases of homologous gene targeting in C.elegans have been reported, no practical technique of gene targeting has been developed so far. In this work we demonstrate that transformation of C.elegans by microparticle bombardment (biolistic transformation) can result in homologous recombination between introduced DNA and the chromosomal locus. We describe a scaled up version of biolistic transformation that can be used as a method for homologous gene targeting in the worm.
Figures
Figure 1
Fig. 1. Ends-in targeting of unc-22 gene. (A) Vector design. Genomic sequence of unc-22 is shown in light gray, homologous sequence included in the targeting vector is dark gray. Two-sided arrows indicate PCR fragments amplified with vector-specific primers B and C and _unc-22_-specific primers A and D located outside the regions of homology. Bold lines below AflII sites indicate positions of a probe for Southern blot analysis, and expected fragment lengths are shown below the probes. (B) Southern blot of unc-22 homologous recombinant (lanes 1–3) and unc-119(ed3) (lane 4) strains. (C) PCR verification of homologous recombination events. Lanes 1–3, independent unc-22 homologous recombinant strains; AB, CD, combination of primers (see A); lane 4, negative control on unc-22 ends-in nonhomologous integrated line; M, marker line.
Figure 2
Fig. 2. Ends-in targeting of unc-54 gene. (A) Vector design. Genomic sequence of unc-54 is shown in light gray, homologous sequence included in the targeting vector is dark gray. Two-sided arrows indicate the PCR fragment amplified with vector-specific primer B and _unc-54_-specific primer E located outside the region of homology. Bold lines below NcoI sites indicate positions of a probe for Southern blot analysis, and expected fragment lengths are shown below the probes. (B) Southern blot of unc-54 homologous recombinant (lane 1) and unc-119(ed3) (lane 2) strains. (C) PCR verification of homologous recombination events using primers B and E (see panel A). Lanes 1 and 2, homozygous unc-54 recombinant worms (unc-54 phenotype); lanes 3 and 4, heterozygous unc-54 recombinant worms (wild-type phenotype but segregate unc-54 progeny); lanes 5–7, independent non-homologous unc-54 transformants; lane 8, no DNA control; M, marker line.
Figure 3
Fig 3. Ends-out targeting of unc-22 gene. (A) Vector design. Genomic sequence of unc-22 is shown in light gray, homologous sequence included in the targeting vector is dark gray. Two-sided arrows indicate PCR fragments amplified with vector-specific primer B and _unc-22_-specific primer F located outside the region of homology. (B) PCR verification of homologous recombination event using primers B and F (see A). Lane 1, negative control on unc-22 ends-out non-homologous integrated line; lane 2, homologous recombinant line; M, marker line.
Similar articles
- Creation of transgenic lines using microparticle bombardment methods.
Praitis V. Praitis V. Methods Mol Biol. 2006;351:93-107. doi: 10.1385/1-59745-151-7:93. Methods Mol Biol. 2006. PMID: 16988428 Review. - Biolistic transformation of Caenorhabditis elegans.
Isik M, Berezikov E. Isik M, et al. Methods Mol Biol. 2013;940:77-86. doi: 10.1007/978-1-62703-110-3_7. Methods Mol Biol. 2013. PMID: 23104335 - Generation of transgenic C. elegans by biolistic transformation.
Hochbaum D, Ferguson AA, Fisher AL. Hochbaum D, et al. J Vis Exp. 2010 Aug 23;(42):2090. doi: 10.3791/2090. J Vis Exp. 2010. PMID: 20811328 Free PMC article. - Reverse genetic strategies in Caenorhabditis elegans: towards controlled manipulation of the genome.
Baylis HA, Vázquez-Manrique RP. Baylis HA, et al. ScientificWorldJournal. 2011 Jul 7;11:1394-410. doi: 10.1100/tsw.2011.126. ScientificWorldJournal. 2011. PMID: 21789474 Free PMC article. Review. - C. elegans gene transformation by microparticle bombardment.
Schweinsberg PJ, Grant BD. Schweinsberg PJ, et al. WormBook. 2013 Dec 30:1-10. doi: 10.1895/wormbook.1.166.1. WormBook. 2013. PMID: 24395815 Free PMC article. Review. No abstract available.
Cited by
- Downregulation of transposable elements extends lifespan in Caenorhabditis elegans.
Sturm Á, Saskői É, Hotzi B, Tarnóci A, Barna J, Bodnár F, Sharma H, Kovács T, Ari E, Weinhardt N, Kerepesi C, Perczel A, Ivics Z, Vellai T. Sturm Á, et al. Nat Commun. 2023 Aug 29;14(1):5278. doi: 10.1038/s41467-023-40957-9. Nat Commun. 2023. PMID: 37644049 Free PMC article. - A Decade of CRISPR-Cas Gnome Editing in C. elegans.
Kim HM, Hong Y, Chen J. Kim HM, et al. Int J Mol Sci. 2022 Dec 14;23(24):15863. doi: 10.3390/ijms232415863. Int J Mol Sci. 2022. PMID: 36555505 Free PMC article. Review. - Animal Models to Study MicroRNA Function.
Pal AS, Kasinski AL. Pal AS, et al. Adv Cancer Res. 2017;135:53-118. doi: 10.1016/bs.acr.2017.06.006. Epub 2017 Aug 8. Adv Cancer Res. 2017. PMID: 28882225 Free PMC article. Review. - Rac1/RhoA antagonism defines cell-to-cell heterogeneity during epidermal morphogenesis in nematodes.
Martin E, Ouellette MH, Jenna S. Martin E, et al. J Cell Biol. 2016 Nov 21;215(4):483-498. doi: 10.1083/jcb.201604015. Epub 2016 Nov 7. J Cell Biol. 2016. PMID: 27821782 Free PMC article. - CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering.
Dickinson DJ, Goldstein B. Dickinson DJ, et al. Genetics. 2016 Mar;202(3):885-901. doi: 10.1534/genetics.115.182162. Genetics. 2016. PMID: 26953268 Free PMC article. Review.
References
- Muller U. (1999) Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech. Dev., 82, 3–21. - PubMed
- Rong Y.S. and Golic,K.G. (2000) Gene targeting by homologous recombination in Drosophila. Science, 288, 2013–2018. - PubMed
- Anderson P. (1995) Mutagenesis. In Epstein,H.F. and Shakes,D. (eds), Caenorhabditis elegans: Modern Biological Analysis of an Organism. Academic Press, San Diego, CA, pp. 31–58.
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources