3. The protocol assumes a GAL4 driver stock in which GAL80
ts
has been included to allow for a temperature shift regime that
restricts knockdown to the 2 days of third-instar development.
This avoids embryonic/larval lethality for genes with essential
roles in early development. The alternative is to carry out the
entire cross at 29
C, which will achieve maximum knockdown
and may be more appropriate for some RNAi stocks. In past
screens, we have found it useful to carry out crosses using both
temperature regimes, in parallel.
4. Test a new driver stock with UAS positive control stocks that
should block eversion. For example, stocks that inhibit the JNK
pathway, such as the RNAi line UAS-bsk.IR, or the dominant
negative UAS-bsk
DN (BDSC#1386), UAS-Timp, to block the
action of MMPs, or the UAS-netA.IR line (VDRC#108577).
Test the GAL4 driver with and without UAS-Dicer-2 and with
and without the GAL80
ts . Dicer-2 can enhance the strength of
the RNAi effect [23]—though this is only of value when using
long hairpin RNAi libraries.
5. The number of pairs in the cross is dependent upon the food.
With the rich molasses-based media we use, five females provide enough larvae to strongly establish the vial (i.e., churn up
the top layer of food) after a day of egg-laying. We recommend
using females for the GAL4 driver, both for the convenience of
collecting virgins of only one genotype, but also because we
tend to find stronger phenotypes when using female drivers.
6. With this regime a vial will be shifted to 29
C when the
progeny is between 2 and 3 days old, which at 25
C corresponds to larvae just beginning second instar to larvae just
beginning third instar. Thus, development will proceed at
29
C for at least the entirety of the third instar. Holding the
crossed parents at the slightly lower temperature of room
temperature will mean the period at 29
C is slightly longer.
We restrict the egg lay to 1 day, so that the number of days over
which flies reach adulthood is minimized to decrease the likelihood that one will stop scoring too soon. It is important that all
progeny that are capable of reaching adult stages are counted
since the more slowly developing progeny often have the stronger phenotypes. We turn adults three times so as to maximize
the n-value for one cross.
7. It is common for a cross to produce only pharate adults. Since
the effects of pEMT failure can be just as easily scored in
pharate adults as eclosed adults, we do not concern ourselves
with the difference. The wings of pharate adults are uninflated
but it is still easy to determine if they are normal or affected
(e.g., Fig. 2g).
130
Sofia Golenkina et al.
ts
has been included to allow for a temperature shift regime that
restricts knockdown to the 2 days of third-instar development.
This avoids embryonic/larval lethality for genes with essential
roles in early development. The alternative is to carry out the
entire cross at 29
C, which will achieve maximum knockdown
and may be more appropriate for some RNAi stocks. In past
screens, we have found it useful to carry out crosses using both
temperature regimes, in parallel.
4. Test a new driver stock with UAS positive control stocks that
should block eversion. For example, stocks that inhibit the JNK
pathway, such as the RNAi line UAS-bsk.IR, or the dominant
negative UAS-bsk
DN (BDSC#1386), UAS-Timp, to block the
action of MMPs, or the UAS-netA.IR line (VDRC#108577).
Test the GAL4 driver with and without UAS-Dicer-2 and with
and without the GAL80
ts . Dicer-2 can enhance the strength of
the RNAi effect [23]—though this is only of value when using
long hairpin RNAi libraries.
5. The number of pairs in the cross is dependent upon the food.
With the rich molasses-based media we use, five females provide enough larvae to strongly establish the vial (i.e., churn up
the top layer of food) after a day of egg-laying. We recommend
using females for the GAL4 driver, both for the convenience of
collecting virgins of only one genotype, but also because we
tend to find stronger phenotypes when using female drivers.
6. With this regime a vial will be shifted to 29
C when the
progeny is between 2 and 3 days old, which at 25
C corresponds to larvae just beginning second instar to larvae just
beginning third instar. Thus, development will proceed at
29
C for at least the entirety of the third instar. Holding the
crossed parents at the slightly lower temperature of room
temperature will mean the period at 29
C is slightly longer.
We restrict the egg lay to 1 day, so that the number of days over
which flies reach adulthood is minimized to decrease the likelihood that one will stop scoring too soon. It is important that all
progeny that are capable of reaching adult stages are counted
since the more slowly developing progeny often have the stronger phenotypes. We turn adults three times so as to maximize
the n-value for one cross.
7. It is common for a cross to produce only pharate adults. Since
the effects of pEMT failure can be just as easily scored in
pharate adults as eclosed adults, we do not concern ourselves
with the difference. The wings of pharate adults are uninflated
but it is still easy to determine if they are normal or affected
(e.g., Fig. 2g).
130
Sofia Golenkina et al.