This week managed to capture the highs and lows of research.
On Tuesday my PCR suggested that I had an ideal mutant for "Project4". Basically the P-element gives the fly red eyes and when the P-element leaves the fly has white eyes. Sometimes when the P-element leaves it takes neighbouring DNA with it and can create mutants. The process is random and has to be muscularly confirmed. I began by screening the lethal candidates, as they are most likely to have a mutation - what with them suddenly being dead. One of the candidates looked like this;
What the hell? So much for that theory of it only deleting in one direction. This is bad news as I have no idea whether the lethality is due to a mutation in "project 4" or "not that way!!!" gene. I'd have to put "not that way!!!" back into this mutant to show the effect was due to loss of "project 4" - if that is even the case. "not that way" gene is a polycomb related gene so it's easy to imagine it could be essential.
This pissed me off quite a bit as it's the second really iffy P-element excision I've had in this lab. The other mutant I've made for "project 2" is what I like to call the "quantum mutant" as it can only be observed indirectly. Basically the P-element is still there when tested indirectly but isn't there when I test it directly. "Project 2" and "other way please" gene are intact but the line is now lethal. The original P-element line is viable...
At this stage I wanted to knock all my fly stocks off the table in a "Bill Adama, wrecking his model ship/painting" rage. The only mutant I've made that makes any sense is viable and it's hard to drum up excitement for a gene that doesn't appear to do anything. A similar thing happened in my previous lab but I was at least able to confirm it mutated the gene I was interested in. Why does this keep happening?. A little bit of investigating revealed that all of the dodgy excisions I've made are ones that use the "Epgy2" P-element. It seems this element takes a perverse joy in screwing you over with confusing results. It also seems to have a tendency to lose the white gene without losing the P-element, so beware if you are doing an excision with it.
Honestly, I think it's time i try some of the new mutant making techniques. Not recombinational knockout as that seems like a ball-ache too. TALENs seems to have potential.
And that's the bad news for the week. I'll leave you on the cliffhanger of the good news until tomorrow (because I have stuff to do now).