Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 83

we performed MosTIC experiments in mutant backgrounds for lig-4 and cku-80,two
genes that are highly conserved and encode factors that are required for nonhomol-
ogous end-joining ( Robert and Bessereau, 2007 ). We demonstrated that in the soma
of the tested mutants, nonhomologous end-joining was defective to repair a Mos1-
excision induced DSB. However, in the germ line, we were not able to detect any
quantitative or qualitative differences between the repair events generated in wild-
type or lig-4 and cku-80 mutant backgrounds.

7. Does Spontaneous Excision of Mos1 Occur?
Yes, rarely. We indeed had noticed that some Mos1 insertions are not stable in the
germ line and can be lost. For this reason, we recommend to check by PCR that the
Mos1 insertion of interest is still present before starting the injection procedure.
Similarly, this can also be done when thawing a Mos1 insertion containing strain,
when a Mos1 insertion containing strain has been maintained for a long time or when
it is difficult to identify a MosTIC-engineered allele.

IV. Materials

A. Equipment

Standard C. elegans culture facility
Standard germ line microinjection setup ( Evans, 2006; Mello and Fire, 1995 )
Dissecting scope equipped for fluorescence detection
Gradient thermal Cycler
Standard setup for agarose gel electrophoresis
Multichannel pipettes
Repeating dispenser

B. Solutions and Reagents
M9 buffer: 22 mM KH 2 PO 4 ,22mMNa 2 HPO 4 , 85 mM NaCl, 1 mM MgSO 4
Worm lysis buffer: 50 mMKCl, 10 mMTris pH 8.2, 25 mMMgCl 2 , 0.45%NP-40,
0.45% Tween-20, 0.01% Gelatin complemented with Proteinase K (final concen-
tration: 1 mg/mL; Eurobio GEXPRK00-6R) before performing lysis.
Phusion high-fidelity DNA Polymerase (Finnzymes, Cat#F-530)
Taq DNA Polymerase (New England Biolabs, Cat#M0273)
Qiaquick gel extraction kit (Qiagen, Cat#28704)
Zero Blunt PCR Cloning Kit (Invitrogen, Cat#K2700)
QuickChange II Site-directed Mutagenesis kit (Stratagene, Cat#200523)

C. Plasmids

The plasmids pJL44 and pJL43 that contain the Mos transposase ( Bessereau et al.,
2001 ) and pPD118.33 (Pmyo-2::GFP) are available at addgene ( http://www.

addgene.org ) .