Saturday, 23 February 2013

This week in the lab (part 1)

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;

All well and good as P-elements are only supposed to excise in one direction meaning "not that way" gene should be intact. However, I decided I should check this, just in case, and because i also wanted a stretch of DNA that covered the mutation for sequencing. This is what happened;

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).


  1. Ohhh the joys of P-element excisiom.... Talens have been working well for me, but can be a pain to construct. If you're not ordering them from somewhere, maybe think about using the cripr/cas9 system. Much easier to get started, though not sure if it has been tested in flies.
    Big hug,

    1. Someone at work was talking about the cas9 system. It sounds like a dream but she was using it in cell lines. I suspect it could be a faff in flies. Will have to keep an eye out for fly version.
      Maybe I should come and visit the UCSF to be bought Talens by an expert :P

  2. The cas9 seems to be working in fish, I'm trying mine next week. And no, even being at Stanford I had to make my own Talens... but would be great to have you visit. Music scene is pretty good here too! What are you waiting for?

    1. Payday/a conference on West Coast of America :)
      Drosophila conference 2014 is in San Diego - there's a goal for me!