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Targeted gene alteration in Caenorhabditis elegans by gene conversion

Nature Genetics volume 36, pages 1231–1237 (2004)Cite this article

Abstract

Now that some genomes have been completely sequenced, the ability to direct specific mutations into genomes is particularly desirable. Here we present a method to create mutations in the Caenorhabditis elegans genome efficiently through transgene-directed, transposon-mediated gene conversion. Engineered deletions targeted into two genes show that the frequency of obtaining the desired mutation was higher using this approach than using standard transposon insertion-deletion approaches. We also targeted an engineered green fluorescent protein insertion-replacement cassette to one of these genes, thereby confirming that custom alleles of different types can be created in vitro to make the corresponding mutations in vivo. This approach should also be applicable to heterologous transposons in C. elegans and other organisms, including vertebrates.

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Figure 1: General strategy for targeted alteration by gene conversion.
Figure 2: Genomic structure of targeted genes.
Figure 3: Gene conversion of a deletion in tkr-1.
Figure 4: Gene conversion of a deletion in frm-3.
Figure 5: Gene conversion of an insertion in frm-3.

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Acknowledgements

We thank J. Collins, C. Almeida, Q. Boese and the other members of the laboratory of J. Collins for sharing unpublished results and providing the TW404 strain; R. Plasterk for his advice and effort in isolating the tkr-1 (pk69) allele; L. Ségalat for providing the frm-3 (cxP4618) allele; C. Gerard for support during initial stages of the project; E. Schwarz for help with Wormbase; A. Fire and M. Han for plasmids; T. Stiernagle and the Caenorhabditis Genetics Center for strains; the Sanger Centre for cosmids; and H. Weinstein for continued support. This work was partially supported by a grant from the US National Institutes of Health to V.G.

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  1. Laboratory of Developmental Immunology, Massachusetts General Hospital, and Department of Pediatrics,, Harvard Medical School, Boston, 02114, Massachusetts, USA

    Peter L Barrett, John T Fleming & Verena Göbel

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Supplementary information

Supplementary Fig. 1

Original PCR gel of first screening round in pilot study. (PDF 73 kb)

Supplementary Fig. 2

Southern hybridization analysis of N2 and tkr-1 (qa100). (PDF 69 kb)

Supplementary Fig. 3

Southern hybridization analysis of N2, frm-3 (cxP4618), (fg2) and (fg3) (frm-3 probe). (PDF 40 kb)

Supplementary Fig. 4

Southern hybridization analysis of N2, frm-3 (cxP4618), (fg2) and (fg3) (GFP probe). (PDF 222 kb)

Supplementary Methods (PDF 58 kb)

Supplementary Video 1

(1) Wild-type (N2), (2) frm-3 (fg2) and (3) frm-3 (fg2); Ex[pfrm-3A + pTG96] animals. Video captures were taken at benchtop conditions using a Nikon SMZ-2B stereomicroscope and Nikon Coolpix 950 digital camera, connected to a Macintosh computer running the program BTV Pro. Apple's iMovie program was used to edit the video. (MOV 59 kb)

Supplementary Video 2

(1) Wild-type (N2), (2) frm-3 (fg2) and (3) frm-3 (fg2); Ex[pfrm-3A + pTG96] animals. Video captures were taken at benchtop conditions using a Nikon SMZ-2B stereomicroscope and Nikon Coolpix 950 digital camera, connected to a Macintosh computer running the program BTV Pro. Apple's iMovie program was used to edit the video. (MOV 172 kb)

Supplementary Video 3

(1) Wild-type (N2), (2) frm-3 (fg2) and (3) frm-3 (fg2); Ex[pfrm-3A + pTG96] animals. Video captures were taken at benchtop conditions using a Nikon SMZ-2B stereomicroscope and Nikon Coolpix 950 digital camera, connected to a Macintosh computer running the program BTV Pro. Apple's iMovie program was used to edit the video. (MOV 113 kb)

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Barrett, P., Fleming, J. & Göbel, V. Targeted gene alteration in Caenorhabditis elegans by gene conversion. Nat Genet 36, 1231–1237 (2004). https://doi.org/10.1038/ng1459

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