1. Targeted gene alteration in Caenorhabditis elegans by gene conversion Peter L Barrett, John T Fleming & Verena Göbel Nat Genet. 2004 Oct 24

2. Present methods for isolating mutations in specific gene in C. elegans using transposon insertions – at least 8 distinct transposons have been identified in C. elegans; mutator strains with ~ 400 times higher efficiency than wild type using chemical- or radiation-based mutagenesis Both methods use PCR for the gene-specific detection of deletions – the location and size of a deletion can be controlled only imprecisely by the selection of the primers – many worms have to be tested Homologous recombination occur only rarely in C. elegans - is not yet used routinely.

3. Strategy for targeted gene alteration by gene conversion – a way to create an engineered deletion in the gene Transposon excision  double-stranded DNA (dsDNA) breaks, which are thought to be repaired in a template-directed manner by means of the sister strand A transgene can also act as a template for repair after the excision of a Tc1 transposon in C. elegans A transgene containing an engineered deletion of a specific size in the genomic DNA corresponding to the area of the Tc1 insertion site can be used as template for repair

5. Isolation of targeted alleles 2 different genes containing Tc1 transposons: tkr-1: conversion plasmid contained a 0.85 kb deletion frm-3: conversion plasmid contains a 1.5 kb deletion Generation of transgenic lines containing the respective Tc1 alleles and conversion plasmids; rol-6 and sur-5::GFP as markers. tkr-1 was tested in mut-2 mutator background frm-3 was tested in mut-2 and mut-7 backgrounds 5-10 parent worms  population of ~ 500 – 1,000 worms Isolation of DNA from about 1/3 of population  Using gene-specific primer pairs to amplify only the gene-converted product, not the transgene

6. Frequency of gene conversion: pilot study with 16 populations tested: 2 positives  2 in 5,333 (much higher than ~ 1 in 100,000 previously reported for point mutations) Confirmation of deletion by sequencing, Southern blots and negative PCR results with primers matching sequences inside the deletion Absence of transgene: loss of roller or GFP and inability to amplify transgene vector sequences from strains tkr-1

7. frm-3

8. Frequencies in different genetic backgrounds Next Comparison of 3 independently derived transgenic mut-7 strains carrying the frm- 3 Tc1 and the conversion plasmid Strains are different in viability transgene copy number and transmission rate 45 populations of each strain were assayed  Similarly high results of 1-3 out of 45 populations  health of the strains the resulting number of generations needed to populate the plate and the properties of the array are not essential for obtaining gene conversion Frequency in mut-2 background was 3 times higher than in mut-7 Same frequency of tkr-1 (0.85 kb deletion) and frm-3 (1.5 kb deletion) in mut-2 background

9. Generation insertion-replacement alleles

11. Advantages of this method High frequencies No screening of large numbers of worms – one to three orders of magnitude lower than in previous screening methods generating custom alleles GFP insertions allowing examination of gene expression in single copy number, in its native genomic milieu and under physiological conditions