Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 79

function of the target gene will make more difficult the identification of MosTIC-
engineered animals either because it will mask the presence of MosTIC-engineered
animals or because it will generate many false-positives.
It should also be kept in mind that DSB repair can sometimes regenerate func-
tional alleles after excision of mutagenic Mos1 insertions with the same efficiency as
transgene-instructed gene conversion ( Robert and Bessereau, 2007; Robert et al.,
2008 and unpublished data) and is also able to generate mutant alleles after excision
of nonmutagenic Mos1 insertions. Hence, it will always be necessary to analyze the
molecular structure of the revertant or mutant alleles selected on individual pheno-
types to identify bona fide MosTIC-engineered strains.
E. An Alternative Protocol Based on the Constitutive Expression of the Mos Transposase
An alternative MosTIC protocol is being developed in our laboratory ( Fig. 5 ). It is
based on the observation that Mos1 insertions can apparently be remobilized directly
in the germ line of animals that have been injected with a vector containing the Mos
transposase under the control of the constitutive germ line promoter Pglh-2
( Frokjaer-Jensen et al., 2008 ).
In this protocol, a DNA mix containing the repair template (50 ng/ m L), the
pJL43.1 plasmid (Pglh-2::MosTase at 50 ng/ m L), and the pPD118.33 (Pmyo-2::
GFP at 5 ng/ m L) is injected into the germ line syncitium of worms homozygous for
the insertion to be remobilized. Each injected P0 animal is kept at 20 C on a NGM
plate and screened for the presence of transgenic worms expressing GFP in the
pharynx in their F1 progeny to verify that they were successfully injected. When
using a phenotypic screening strategy, candidate MosTIC alleles can be screened in
the progeny of P0 animals that segregate transgenic animals. When using PCR
screening, consider each P0 plate containing F1 progeny as a pool and use the
sibling-selection strategy described previously.
Interestingly, we observed that MosTIC alleles could arise at later generations
using Pglh-2::MosTase. Therefore, it is worth isolating transgenic lines and screen-
ing for MosTIC alleles at subsequent generations if no MosTIC-engineered allele is
recovered on the P0 plates. To minimize germ line silencing of the extrachromo-
somal array containing the Mos transposase source, we recommend to maintain the
transgenic lines at 25 C.
Using this protocol, we constructed three independent KI alleles of unc-29, a gene
coding for a subunit of the acetylcholine receptor expressed at neuromuscular
junctions. Based on phenotypic screening, we were able to recover MosTIC-engi-
neered alleles with a frequency ranging from one MosTIC-engineered allele out of
7 successfully injected animals (14%) to one MosTIC-engineered allele out of 15
successfully injected animals (6.6%) ( Fig. 5 B).
When using a phenotypic screening strategy, engineered alleles can be obtained in
less than 10 days after injection. When using a PCR-based strategy, engineered
alleles are recovered in 3 weeks after injection.