Jun
02

AP Axis Neural Plasticity-1

I came back to campus a week early so I could get a head start on my project. Currently, I’ve been trying to clone a list of candidate genes from total Xenopus laevis cDNA that are differentially expressed between sham and rotated transplants (refer to my intro post for more detail. I will then synthesize RNA probes from these clones for ISH. The following are my progress so far:

Plasmid clones made for:

Harbi1, EGFL6, Six3, Gsx1, Prdm13, Ptbp3, Trpv2, Prom1, Tex36, Ptf1a, EGFL6 (probe), Atoh7

ISH Probes made for:

Harbi1, EGFL6, Six3, Gsx1, Prdm13, Ptbp3, Trpv2

Probe test ISH done for:

EGFL6:

Twice. Expression was visible only for stage 30 or older embryos, at dorsal and ventral tail fin area. Since there is no visible expression at stage 18, when EGFL6 is DE between Sham and Rotated embryos, we decided against performing EGFL6 ISH on transplant embryos. However, this lack of signal in earlier stage embryos might be attributed to the fact that our probe is specific for only the S homeolog, and is much longer than 1kb, the conventional maximum probe length. That’s why a new probe that is much shorter and could detect both S and L homeolog mRNA was cloned.

Harbi1:

Once. Both S and AS embryos produced significant and identical background, probe test not successful. Will use embryos fixed by myself next time to try again, since my advisor said the embryos looked “under fixed”.

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