Frog Prrx1:CreER line screening

Transgenic TLM115 Frog Screening Protocol 2022.07.18 Tzi-Yang

A. Mating (ask Birgit if not clear), ~1 hour

• Choose the mating pairs and make notes on the frog list.
  • E.g. TLM115_F_02 x C36_107M_201M
• Thaw hCG stored in the -20 degree freezer (Xenopus drawer).
• Bring 20L tank(s) and lids in the frog exp room with a trolley (1 tank per mating pair).
• Prepare 7-8L 20mM NaCl per tank:
  • 5M NaCl 26uL per tank
  • Add tap water to 7-8L mark on the tank (or roughly 2/5 to 1/2 of the tank)
• Find animals in the colony using the chip reader or photos.
• Put the pairs together in a tank and label the tank.
• Inject hormones and put the pairs back to the tank:
  • 400-500unit hCG per female
  • 40-50unit hCG per male
• Cover the tank with a carton box or an orange bag.

(Leave the tank on the trolley at the corner of the frog exp room overnight.

B. Collecting eggs (The next day afternoon), ~1-2hr

• Print labels.
• Collect the eggs to a white box with tap water.
• If the egg clumps together, separate them into smaller clumps.
  • Dejellying and picking the surviving embryos is recommended.
  • Collect ~500 embryos in 2 boxes would be enough.
• Change water at least every other day!

C. First Screening at 7-8 days after mating. ~1-2 hours

• • The timing is important because 1) the embryos should be floating but not active swimming at these stages, so we do not need to anesthetize them. 2) and it is the earliest timing when the trunk-GFP and lens-Cherry signals could be easily distinguished.
• First screen for trunk-GFP+ embryos using a plastic dropper under the fluorescence stereoscope.
• Then screen for lens-mCherry+ embryos in the trunk-GFP+ embryos.
• Grow the double positive animals and label +/+ on the white box.
  • Start feeding only from 14 days or later. Feeding too early and feeding too much will kill them.

D. 4OHT treatment: ~15-25 days after mating, ~30min/per treatment

• Thaw a tube of 4OHT stock (20mM in DMSO) in dark.
• Prepare 1.5uM 4OHT in 0.1xMMR in a genotyping box (200mL per ~100 animals)
  • 4OHT (20mM) 15uL
  • 10xMMR 2mL
  • MilliQ water to 200mL
• Transfer the embryos into the 4OHT solution. Keep the box in dark overnight.
• (The next day, ~18-22 hours later) Wash and transfer the animals to tap water.
  • Feed and clean daily until the next treatment.
  • To get the best conversion result, do 2x treatments per week for two weeks.
  • Do not put them into the system during the treatment period. Wait at least 5-7 days from the last treatment.

E. Second screening: >7 days after the last treatment, or until stage >52 (~2 month old).

• Anesthetize the animals briefly
• Screen for Cherry+ limbs under the fluorescence stereoscope.

• Grow the converted animals.
• Label the tank and the frog list properly about the result of 4OHT treatment.
  • The best screening stage is between stage 51-53.
  • The Cherry signal could be observed in the limb mesenchyme and the mid brain regions. Occasionally the Cherry signal could be found in tail muscle.
  • IMPORTANT: there will be ~ 30% of the double positive animals that do not have conversion in the limb. The reason is yet to be determined. So always screen properly!
• Grow the converted animals up and use them for experiments.

F. (Optional) Third screening: stage 66 (~ 6-7months old)

• Keep the animals with strong conversion and clean conversion result for the next generation. Strong conversion means the Cherry signals are strong in the joints. Clean conversion means no muscle were labeled in the froglet leg. Below is an example.