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An example of a setup experiment is shown
Fig. 4
B. It demonstrates that
jumping PCR is detected when primer annealing is performed between 60
C
and 66.4
C. There is no PCR jumping when primer annealing is performed at
68.4
C. However, the control plasmid can still be amplified using this latter
annealing temperature suggesting that a MosTIC allele with a similar structure
should be detected under these conditions. A similar experiment was performed
on pools of heat-shocked transgenic animals derived from the lines tested on
Fig. 4B (Fig. 4C)
, five pools out of six were positive when annealing the primers
at 64
C. In contrast, only pool 3 was still positive when annealing the primers at
68.4
C. Further screening confirmed that pool 3 indeed contained a MosTIC-
engineered allele.
(c) PCR Screening Using a Sibling-Selection Strategy
1. Wait for the pools of heat-shocked animals (cf. Part III. B) to exhaust their
food supply.
2. Wash half of the plate with 1 mL of M9 1
buffer and transfer the animals to
a 1.5 mL tube. Let the worms sediment on ice, collect 50
m
L at the bottom of
the tube, and transfer the worms to PCR plates.
3. Perform lysis and PCR as previously described (cf. Part III. C. 1. b 2 & 3)
using the optimized PCR parameters.
4. Once a positive PCR signal is identified, transfer a chunk of the correspond-
ing plate to a fresh plate. From the developing population, make 15 pools of
20 nontransgenic (non-GFP-positive) animals. Selecting nontransgenic ani-
mals at this step (i) prevents potential jumping PCR problems and (ii) does
not affect the recovery frequency of MosTIC events since they happened in
the germ line of the heat-shocked animals few generations before. Wait for
the pools to exhaust their food supply and analyze them as described above.
At this step, MosTIC-engineered alleles can be, most of the time, detected by
a single PCR round. For no clear reason, we sometimes observe a significant
decrease of the efficiency of the nested PCR. It might be due to the absence of
single-strand DNA generated from the transgene and that is used as PCR
template in the previous screening step.
5. From one positive subpool, clone 40-80 individuals to single plates to
identify the MosTIC-engineered strain.
2. Phenotypic Screening
When phenotypic screening strategies could be used, they turned out to be very
efficient in some of our experiments (
Robert and Bessereau, 2007
and unpublished
data). They can be used, for instance, to generate a KO/del allele starting from a
nonmutagenic Mos1 insertion or to generate a functional KI allele starting from a
mutagenic Mos1 insertion. In both cases, it is recommended to work with nonrescu-
ing repair templates. The presence of a repair template able to rescue by itself the
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