Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 75

C. Heat-Shock Induction of Mos1 Excision and Pooling of Heat-Shocked Animals
(a) Select nematode growth medium (NGM) plates ( Stiernagle, 2006 ) containing
about 200 young transgenic adults from one to five transgenic lines, seal with
parafilm and immerse for 1 h in a water bath setup at 33 C.
(b) Let the worms recover for 1 h at 15 C.
(c) Immerse for 1 h in a water bath setup at 33 C. Remove the parafilm.
(d) After one night at 20 C, transfer heat-shocked animals to fresh plates.
Depending on the fertility of the heat-shocked transgenic animals, put one to
five animals on the same plate in order to obtain 100 F1 animals in each pool. To
calibrate this step, we recommend to heat-shock few transgenic animals and
estimate their brood size before starting the MosTIC experiment.
(e) Before screening, roughly estimate the F1 population size to be able later to
calculate the MosTIC efficiency.
Typically, 50-100 pools are screened for a single heat-shock induction experi-
ment. If MosTIC occurs with a frequency of 5 10 -4 events per offspring in the
progeny of the heat-shocked animals, the probability of recovering at least one
MosTIC allele when screening 10,000 progeny of heat-shocked animals is 99%. If
MosTIC efficiency is lower, we recommend to repeat several heat-shock induction
experiments of the same size rather than increasing the size of a single induction
experiment.
D. Screening
1. PCR Screening
In most cases, identification of MosTIC KO/del and KI alleles relies on PCR
screening, which is performed in two successive rounds of nested PCR. To facilitate
the handling of a large amount of samples, we recommend towork with multichannel
pipettes and repeating dispenser using 96-well PCR plates.
(a) Primer Design and ''Jumping PCR'' ( Fig. 4 )
For each primer pair, one primer is located inside the repair template and the
other one outside. For KO/del alleles, the ''inside'' primers recognize the repair
template on its left/long arm (primers P5 and P7 on Fig. 3 ). For KI alleles, the
''inside'' primers recognize the repair template in the tag sequence (primers P7 0
and P9 0 on Fig. 3 ). In both case, the ''outside'' primers recognize genomic
sequences to the right of the short arm.
One common pitfall of this strategy arises from PCR fragments having the same
size as the specific PCR product amplified in the presence of a MosTIC-engi-
neered allele and sometimes amplified from transgenic animals that do not
contain MosTIC-engineered alleles ( Fig. 4 A). This artifact arises by ''jumping
PCR'' ( Paabo et al., 1990 ) between single-stranded DNA generated from the
transgene on the one hand and the genome on the other hand ( Fig. 4A and B ).
Jumping PCR is dependent on the nucleotidic composition of the amplified