Christelle is on vacation in Japan. This is our last phone call catching up, just before she left at the end of July.
- ISL1 is not expressed in HeLa cells (it is in 293 cells). Transfect truncated ISL1 construction and ISL1-Myc construction into HeLa cells. The truncated one is because the N-terminal LIM domain seems to fold over and repress DNA binding to the homeodomain unless the LIM is otherwise engaged; C-terminal to that is an activation domain to get translocation into the nucleus, but the LIM domain probably doesn’t act on that. So the truncated version has no LIM domain on it.
- First Christelle did a Western with an anti-Myc and got highly positive signal not in cytoplasmic fraction but in the nuclear fraction, as hoped. Not sure if the ISL1 antibody itself would work in WB. Turned out ok (does that mean it did?) with a 1/10 dilution.
- She did an EMSA with a control oligo that would work for ISL1 binding, within the ISL1 promoter itself. This oligo is chemiluminescent because it has incorporated biotin on one end (ordered it biotinylated rather than making since only one oligo). Gets a good shift when add in expressed protein mix, 20 ug of which she can isolate from her transfected HeLa cells. The reticulocyte supernatant (?) bound the oligo non specifically so had to no longer use that system. When get the shift can compete it off with with non-biotin-labeled oligo at 1/200 dilution.
- Now developing oligos with the 6nt consensus sites mutated to random nt instead of the canonical sequence.
- Developing constructions to go into the Dual luciferase assay, since these elements are far away from what is probably the real FGF promoter whose activity we’d like to see regulated by ISL1. Choosing, if I remember, a flanking sequence of some 50-odd nt? But I didn’t write this down.
- FGF ISH again did not work on the C18 embryo. However, the immuno did, giving a signal in the diencephalon as expected and I should have tried to see those slides but forgot during my early August visit.
- Also did additional HIS in the RET+ enteric plexus of a postnatal child provided by F. Joubert, also saw it was expressed in the colonic crypts. I integrated these photos into a figure for Loic’s article submission.
- Alethea 11:12, 9 August 2008 (UTC):