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. tkr-1 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

7. frm-3

8. Frequencies in different genetic backgrounds • 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