Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 81

(7 days), progeny of the injected animals are screened for MosTIC-engineered KO/
del alleles by looking at the worms that are rescued for unc-119 but that do not
express any of the injection markers. Most of them contain a MosTIC-engineered
allele in which the region to delete has been replaced by the unc-119 marker.
Using this protocol, it has been possible to (i) generate several deletions at the
dpy-13 locus, some of them being 25-kb long, (ii) delete the tandem gene duplica-
tion containing the cst-1 and cst-2 genes, and (iii) delete the essential gene dyn-1.
Interestingly, the presence of the unc-119(+) marker at the site of the deletion makes
a perfectly balanced lethal chromosome. A detailed protocol is provided in Frokjaer-
Jensen et al. (2010).
This method seems to be extremely simple and powerful to select MosTIC-
engineered KO/del alleles without using PCR screening. It remains, however, to
evaluate the potential effect of the unc-119(+) fragment on the expression of nearby
genes.
G. Frequently Asked Questions
1. When is it Better to use the Alternative MosTIC Protocol Based on Constitutive Germ Line
Expression of the Mos Transposase Instead of the Standard Protocol?
The standard MosTIC protocol, requires the microinjection of only few animals
containing the Mos1 insertion of interest in order to generate transgenic lines. The
alternative protocol requires more time microinjecting but permits the identification
of engineered alleles a few days after injection. However, the overall MosTIC
efficiency depends on the quality of the injection, which varies with the researcher
injection ability and the genetic background. If the MosTIC events are predicted to
be rare (engineering site far from the Mos1 insertion), we recommend to use a heat-
shock-based strategy.
2. What is the Optimal Length for the Homologous Arms Present on the Repair Template?
Standard repair templates contain two 1.5 kb regions of homology (see Part III.A.1
for details). We tried to increase the length of one of this arm but did not observe a
significant increase in MosTIC efficiency ( Robert and Bessereau, 2007 and Fig. 2 B).
On the contrary, shortening one of the arm length down to 700 bp dramatically
decreased MosTIC efficiency.
When designing a repair template, there are two points to keep in mind depend-
ing on the screening methodology. First, if a phenotypic screening strategy is used,
it is essential that the repair template by itself does not rescue the mutant pheno-
type. Second, if screening by PCR, it is essential to keep at least one of the
homologous arm short enough to be able to design primers outside of the repair
template. In our experiments, we usually keep the short homologous arm at 1.5 kb
and we design primers in such a way that PCR fragment will range between 1.8
and2kb.