Download Folie 1 - Department of Zoology, UBC

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 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
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
frm-3
Frequencies in different genetic backgrounds
Comparison of 3 independently derived transgenic mut-7 strains carrying the frm3 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
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