Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 70

engineering avoids overexpression artifacts and provides more physiologically rel-
evant results (see ''Discussion'' for an example). Here, we provide a detailed
description of the MosTIC protocol routinely used in our laboratory and we discuss
an alternative protocol that we have established.
II. General Features of the MosTIC Technique and
Experimental Outline
An overview of the MosTIC procedure is depicted in Fig. 2 A and can be sum-
marized in five points:
(i) A Mos1 insertion is identified in the region of interest.
(ii) A repair template containing the desired modification is designed.
(iii) Transgenic lines containing the selected Mos1 insertion are generated with the
repair template and a vector providing the expression of the Mos transposase
under the control of a heat-shock inducible promoter.
(iv) Mos1 excision and MosTIC are triggered by a heat-shock treatment.
(v) MosTIC-engineered alleles are identified using either PCR or phenotypic
reversion to screen the progeny of heat-shocked animals.
Using this protocol, it is possible to engineer point mutation alleles, KO/del alleles
and KI alleles expressing tagged protein versions ( Fig. 2 B). MosTIC alleles engi-
neered at the Mos1 insertion point are recovered with frequencies ranging from 10 -3
to 10 -5 events per offspring. This frequency decreases when the modification to
introduce into the genome is located further away of the Mos1 insertion point.
Characterization of a MosTIC conversion tract was performed at the unc-5 locus
( Robert and Bessereau, 2007 and Fig. 2 C) using a repair template containing mul-
tiple silent polymorphisms. Point mutations localized in a 1 kb-long fragment
centered at the Mos1 insertion point were copied in at least 50% of the MosTIC
alleles. The recombination efficiency was decreased 20 times 1.5 kb away from the
Mos1 insertion point.
Using this standard protocol, we have been able to generate seven different
MosTIC KI alleles at five independent loci of the C. elegans genome ( Gendrel
et al., 2009; Robert and Bessereau, 2007 ; and V. Robert, T. Boulin, C. Stigloher,
H. Tu and J.L. Bessereau, unpublished results). Tags have been inserted at distances
varying from 20 to 800 bp of the Mos1 insertion point and estimated MosTIC
efficiencies were similar to the ones observed for point mutation engineering.
When it is possible to screen for an engineered allele based on a visible pheno-
type, it takes only 2 weeks after heat-shock induction of the Mos transposase
expression to isolate a strain containing the MosTIC allele. When using a PCR-
based strategy, several rounds of sibling selection are required to isolate single
modified animals and it takes about 1 month after the initial heat-shock to isolate
the MosTIC allele.