Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 85

UNC-63 is detected by immunostaining at synapses in the nerve ring, dorsal nerve
cord, and ventral nerve cord. When expressed from an extrachromosomal array
under the control of its endogenous promoter, UNC-63-YFP rescues the mutant
phenotypes of an unc-63 null but the fusion protein is diffusely distributed in
intracellular muscle compartments and in the plasma membrane. On the contrary,
when the unc-63 locus is engineered by MosTIC, UNC-63-YFP is only present at
synapses and colocalizes with the other subunits of the acetylcholine receptor.
For germ line-expressed genes, experimental evidence are still required but
MosTIC-engineered alleles should be extremely valuable tools since it has been
so far extremely difficult to construct healthy strains expressing tagged versions of
genes expressed in the germ line.
B. Single-Copy Transgene Genomic Integration by MosSCI
Quickly after its establishment, MosTIC protocol was adapted to derive an
efficient technique of single-copy transgene insertion known as MosSCI (for
Mos1-mediated single-copy insertion) (( Frokjaer-Jensen et al., 2008 ) and http://
sites.google.com/site/jorgensenmossci/Home ). Using MosSCI, a transgene can be
inserted as a single-copy at an intergenic position defined by a pre-existing Mos1
insertion. In this case, the repair template is made of the sequence of interest flanked
by two DNA arms, which are homologous to the genomic regions flanking the Mos1
insertion point. After induction of Mos1 excision in the presence of the Mos trans-
posase, positive and negative-counter selections are used to select DSB repair events
resulting in the single-copy integration of the extrachromosomal repair template.
Using MosSCI, single-copy transgene integrations could be recovered at least at two
''neutral'' intergenic positions defined by Mos1 insertions that were chosen because
they apparently do not induce phenotypic changes. MosSCI alleles exhibit a stable
and robust expression both in the germ line and the soma ( Frokjaer-Jensen et al.,
2008 ) and first examples of studies based on the analysis of MosSCI alleles have
been recently published ( Lehrbach et al., 2009; Pagano et al., 2009 ).
VI. Summary
The MosTIC technique described in this chapter provides a way to engineer
customized alleles in C. elegans. It requires an insertion of the Mos1 transposon
in the target locus. Excision of the transposon triggered by germ line expression of
the Mos transposase creates a DNA DSB that locally stimulates homologous recom-
bination. Sequence variations contained in a repair template provided in a transgene
can be copied into the broken locus by gene conversion. In the standard MosTIC
protocol, Mos1 transposition is induced by heat-shocking transgenic lines carrying a
Mos transposase expression vector under the control of a heat-shock inducible
promoter and an engineered repair template. MosTIC alleles can be identified in
the progeny of the heat-shocked animals by PCR or based on the appearance of a new