Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 74

''KI'' repair templates are synthesized in two successive rounds of PCR fusion.
First, primers P1 0 and P2 0 ( Fig. 3 C) are used on wild-type genomic DNA to amplify
the long arm of the repair template and P3 0 and P4 0 are used to amplify the tag
sequence using an appropriate PCR template. P2 0 contains in its 5 0 end a tail of 24
nucleotides, which overlaps with the end of the tag sequence. PCR fusion is per-
formed using primers P1 0 and P4 0 and an equimolar mix of P1 0 -P2 0 and P3 0 -P4 0 PCR
products. The short arm of the repair template is amplified on wild-type genomic
DNA using P5 0 and P6 0 .P5 0 contains in its 5 0 end, a tail of 24 nucleotides, which
overlaps with the end of the tag sequence. PCR fusion is performed by mixing
equimolar quantities of P1 0 -P4 0 and P5 0 -P6 0 PCR products and using primers P1 0
and P6 0 .
Repair templates for point mutations can be constructed either by PCR fusion-
based strategy with primers containing the mutations or by site-directed mutagenesis
of a subcloned genomic fragment using, for example, the QuickChange II kit
(Stratagene).
Several mechanisms are at work in the C. elegans germ line to repair a Mos1-
triggered DSB ( Robert et al., 2008 ) and sometimes regenerate sequences encoding a
functional protein. When screening MosTIC-engineered alleles by phenotypic rever-
sion, it might be useful to be able to quickly distinguish between revertants generated
by transgene-instructed gene conversion from those generated by other mechanisms.
In that case, we recommend to introduce a silent restriction site in the repair template
close to the DSB site (see Robert and Bessereau, 2007 for an example).
2. Establishment of Transgenic Lines
(a) Use standard C. elegans germ line microinjection procedure ( Evans, 2006;
Mello and Fire, 1995 ) to generate extrachromosomal arrays carrying both the
repair template and a heat-shock inducible Mos transposase expression vector in
a genetic background homozygous for the Mos1 insertion to mobilize.
Into a strain homozygous for the Mos1 insertion of interest, inject the following
mix:
- the repair template at 50 ng/ m L.
- pJL44 (Phsp-16.48::MosTase) at 50 ng/ m L.
- pPD118.33 (Pmyo-2::GFP) at 5 ng/ m L as a transformation marker.
Inject about 20 animals and keep them at 20 C prior to transgenic F1 screening.
(b) Isolate individual F1 transgenic animals with expression of GFP in the pharynx
and screen their progeny to identify transgenic lines. Usually, 10-30% of the F1
transgenic animals give transgenic lines. Select five healthy lines derived from
different F1 transgenic animals, with an intermediate to high transgene trans-
mission rate ( > 50% transmission of the transgenic array).
(c) Amplify the transgenic populations by maintaining the transgenic lines at 25 C,
if possible, in order to minimize potential transgene silencing.