swPCR of SPD-2 F2 and F3

Following my previous blog post, I had screened the F1 generation of the injected worms with SPD-2. I identified that 2 of the 100 worms appeared to be heterozygous for SPD-2, with the insert and WT. From these 2 worms (worm 9 and worm 17), my supervisor had picked 12 worms from each of these plates and plated the F2s individually. We then waited until these F2's laid enough embryos and had enough progeny so they can be screened.

Once the worms were ready, I repeated the screening process on these F2s, with hopes that one of these worms will present a homozygous genome for SPD-2 - containing just the insert. I picked the worms and placed them into 20ul of 1x PCR buffer, froze them in the -80C freezer for 30 minutes, then lysed them using proteinase K, which was subsequently activated in the PCR machine. The lysed worm mix was then added to a PCR mix to amplify SPD-2, and a gel was run to identify whether our worms had the insert or not.

swPCR analysis of F2s from worm 17

Unfortunately, all but one worm presented a homozygous genome for SPD-2, presenting the WT band. This suggests that neither worm 9 or worm 17 actually had the insert in the first place, and can likely be a result of noise.

Worm 9.1 displayed two bands, suggesting the presence of both of insert and WT SPD-2, with a band clearer than its parent, worm 9.

swPCR analysis of F2s from worm 9. Worm 9.1 appears to be heterozygous for the insert.

To follow up from this screening, F3 progeny from worm 9.1 will be screened to see if any of the progeny is homozygous for the insert.

These specific results did leave me very confused as although there was presumably a band present, almost all offspring do not have the insert. However, one did, and if one did have the insert, then it suggests the parent must of have had the insert aswell. Discussing this with my PI, Dr Erik Griffin, we identified possible biological and technical explanations for these results. It could be that the homozygous worms are not liable and have died before even hatching from the embryos, or, as stated before a result from noise.

Following the F3 worms, progeny from 9.1 were screened to check if any of it's offspring were homozygous. Results from this screening showed all worms to have the WT band for SPD-2, and no band for the insert. With no clear explanation now, and just theories that the homozygous worms die, we decided to re-inject the worms with SPD-2, to start again from the F1 generation.

Although being frustrating, I have learnt that this process has a very low efficiency, and as my supervisors say, hope for the best but expect the worst!