Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 72

III. Methods

A. Identification of a Mos1 Insertion in the Target Region
MosTIC requires an entry strain containing a Mos1 insertion preferentially
located at 500 bp or less from the target site. A Mos1 insertion library has been
generated by the Nemagenetag consortium ( Bazopoulou and Tavernarakis, 2009;
Duverger et al., 2007 ). It contains 13,845 independent insertions, which provide
entry points for manipulating about 10% of the genome. Mapped insertions are
annotated in wormbase ( http://www.wormbase.org/ ) andC. elegans stocks car-
rying these insertions are distributed upon request ( http://www.cgmc.univ-lyon1.
fr/cgmc_info_celeganstp.php ). Alternatively, strains containing Mos1 insertions
in a gene of interest can be generated by performing its own Mos1 mutagenesis
( Williams et al., 2005 ) as described in the following protocols ( Bessereau, 2006;
Boulin and Bessereau, 2007 ). Mos1 mutagenesis strategy can be extremely
useful to identify Mos1 insertions in several genes involved in the same biolog-
ical process and further use the Mos1 alleles as entry points for genomic

engineering.

B. Construction of Transgenic Lines
1. Building the Repair Template
Standard repair templates designed to engineer MosTIC KO/del or KI alleles are
described in Fig. 3 . A standard MosTIC repair template ( Fig. 3 A) contains the
modifications to introduce into the genome flanked by one long arm and one short
arm of genomic sequences. The long arm (left arm on Fig. 3 ) contains the genomic
sequence located between the point where modifications have to be introduced in the
locus and the Mos1 insertion point and an additional 1.5-kb long genomic fragment
flanking the left side of the Mos1 insertion point. The short arm (right arm on Fig. 3 )
contains a 1.5-kb long genomic fragment at the right side of the genomic point where
the modifications have to be introduced. The repair template is preferentially made
using PCR fusion-based strategies ( Hobert, 2002 )( Fig. 3 ) but any molecular biology
techniques can be used. Next, it is cloned into a standard plasmid and sequenced over
its full-length before injection.
For the construction of ''KO/del'' repair templates, independent PCR are per-
formed with primers P1-P2 and P3-P4 ( Fig. 3 B) on genomic DNA extracted from
wild-type animals. P2 is 44 bp long and overlaps the region that has to be deleted. Its
3 0 end contains a 20 nucleotide-long sequence complementary to the segment that
flanks the deletion on its left side and its 5 0 end contains a 24 nucleotide-long
sequence complementary to the segment that flanks the deletion on its right side.
This sequence will hybridize with the end of the P3-P4 product during the PCR
fusion reaction, which is performed using primers P1 and P4 and an equimolar mix
of P1-P2 and P3-P4 PCR products. Amplification can be performed using the
Phusion high-fidelity DNA Polymerase (Finnzymes) or any other high-fidelity