Homologous gene targeting in Caenorhabditis elegans by biolistic transformation

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.

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.