Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 66

Abstract

The nematode Caenorhabditis elegans is an anatomically simple metazoan that
has been used over the last 40 years to address an extremely wide range of
biological questions. One major advantage of the C. elegans system is the
possibility to conduct large-scale genetic screens on randomly mutagenized ani-
mals, either looking for a phenotype of interest and subsequently relate the
mutated gene to the biological process under study (''forward genetics''), or
screening for molecular lesions impairing the function of a specific gene and
later analyze the phenotype of the mutant (''reverse genetics''). However, the
nature of the genomic lesion is not controlled in either strategy. Here we describe
a technique to engineer customized mutations in the C. elegans

genome by

homologous recombination.
This technique, called MosTIC (for Mos1 excision induced transgene-
instructed gene conversion), requires a C. elegans strain containing an insertion
of the Drosophila transposon Mos1 within the locus to modify. Expression of the
Mos transposase in the germ line triggers Mos1 excision, which causes a DNA
double strand break (DSB) in the chromosome at the excision site. The DSB
locally stimulates DNA repair by homologous recombination, which can some-
times occur between the chromosome and a transgene containing sequence
homologous to the broken locus. In that case, sequence variations contained in
the repair template will be copied by gene conversion into the genome. Here we
provide a detailed protocol of the MosTIC technique, which can be used to
introduce point mutations and generate knockout and knock-in alleles.

I. Introduction

Gene knockout (KO/del) and knock-in (KI) have emerged as essential com-
ponents of the genetic toolbox used to study gene function in model organisms.
In yeast and mouse, techniques have been developed in the eighties to engineer
chromosomal loci ( Doetschman et al., 1987; Scherer and Davis, 1979; Thomas
and Capecchi, 1987 ); these rely on the recombination between the chromosome
and a transgenic DNA fragment carrying the sequence to introduce into the
genome flanked by sequences homologous to the targeted locus. Positive and
negative selection markers are used to identify recombination events and select
against random insertion into the genome. In the nematode Caenorhabditis
elegans, however, the low frequency of spontaneous recombination between the
genome and exogenous DNA has impeded for a long time the establishment of an
equivalent strategy. Therefore, most of the data generated in C. elegans to
characterize gene expression patterns or to define the subcellular localization
of tagged proteins relied on the use of transgenes made of repetitive extrachro-
mosomal arrays.