Targeted gene alteration in Caenorhabditis elegans by gene conversion

Peter L Barrett, John T Fleming & Verena Göbel

Nat Genet. 2004 Oct 24

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.

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

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 backgroundfrm-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

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

frm-3

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 viabilitytransgene 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 strainsthe 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

Generation insertion-replacement alleles

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