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3. Did you Try to Have the Repair Template and the Mos Transposase on Two Different Arrays?
Why Don ' t you use the Existing hsp::Tpase Array oxEx166 ( Bessereau et al., 2001; Boulin
and Bessereau, 2007; Williams et al., 2005 )?
Initial MosTIC experiments were performed using the existing oxEx166 array,
which contains heat-shock inducible source of Mos transposase, combined with an
independent array carrying the repair template. In this configuration, MosTIC was
not more efficient than using a single array carrying both the transposase and the
repair template. Therefore, we recommend to generate a single extrachromosomal
array because it is sometimes problematic to select and amplify double transgenic
animals. In addition, we think that using a freshly injected source of transposase for
each experiment minimizes the risk of working with transgenes that might have
undergone silencing.
4. Did you Try Other Coinjection Markers than pPD118.33 (Pmyo-2::GFP)?
Any coinjection marker might work as long as it is easy to detect. For example, we
used the dominant injection marker rol-6(su1006) contained on plasmid pRF4
( Mello et al., 1991 ). However, extrachromosomal arrays are not always stable in
mitosis or meiosis. As a consequence, transgenic animals are mosaic, meaning that,
in a single animal, some cells carry the transgene whereas others have lost it
( Stinchcomb et al., 1985 ). Mosaicism can sometimes complicate the discrimination
between transgenic animals from nontransgenic animals, which is an issue when
screening by PCR. In our experiments, confusion between nontransgenic and trans-
genic animals happened when working with pRF4. On the contrary, we did not have
significant problems discriminating between nontransgenic and transgenic animals
when using pPD118.33 as a transformation marker, which we therefore recommend.
5. Is MosTIC More Efficient in Genetic Backgrounds in the Absence of Germ Line Transgene
Silencing?
No. In the C. elegans germ line, extrachromosomal arrays are submitted to silencing
(see ''Introduction''). It might result in decreased transposase expression and Mos1
excision frequency. To test this hypothesis, we performed MosTIC experiments in the
mut-7(pk204) background where germ line transgene silencing is released ( Ketting
et al., 1999; Robert and Bessereau, 2007 ). In this mutant background, MosTIC events
were recovered at the same frequencies as in the wild-type background suggesting that
in spite of transgene silencing, the amount of Mos transposase present in the germ line
was not a restrictive factor for MosTIC efficiency.
6. Is MosTIC More Efficient in the Absence of End-Joining DSB Repair?
Several DSB repair mechanisms, including end-joining, are at work in the
C. elegans germ line, raising the possibility that they might compete with trans-
gene-instructed gene conversion and decrease its efficiency. To test this hypothesis,
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