Every day for more than a month now, first thing in the morning, I've been checking through several hundred newly hatched Drosophila, searching for one with red eyes in among all the white-eyed ones. This is the end of an experiment begun maybe a year ago, possibly a bit less. I started by cloning a piece of DNA from the mosquito genome, then I stuck it into a plasmid (a ring of DNA that can be put into a bacterium), put the plasmid into a bacterium, grew the bacteria and harvested the DNA. I sent the bits off for sequencing, to check they were what they were supposed to be. That would have taken maybe two months, with several tries, because it's a long piece of DNA and awkward to use.
Once I knew I had the right thing, I tried to put the DNA into another plasmid. I tried for a long time, before working out what was wrong (ten very important base pairs were incorrect - 10 in 3000). Then I had to start at the beginning again. After getting to this point with the right bit of DNA, I had to put it into the second plasmid, put that into a bacterium, and grow it up. Then it had to be sequenced, again. That must have taken another couple of months at least, on top of the time it took to begin again. The second plasmid was harder to use than the first.
Once I had the right bit of DNA, the right way round, in the second plasmid, in a bacterium, I grew lots of it and harvested it, precipitated it and added a helper plasmid so it was the right concentration. Then I started injecting it into Drosophila eggs from a strain mutant for the gene white - they have white eyes, not red. I had to grow the few larvae that survived to adults and then cross them with Drosophila which are also mutant for white. Then I had to look at every resulting adult, for one that had red eyes.
The plasmid I put into the eggs carried my bit of DNA stuck in front of a marker gene called lacZ, and also a functioning copy of the white gene, all inside P elements, which are certain short sequences: P-white-my DNA-lacZ-P.
The helper plasmid carried a gene called transposase, which makes a protein that cuts at the P elements and puts them and whatever is inside them elsewhere. By injecting my plasmid and the helper plasmid,I was hoping that the helper plasmid transposase would cut at the P elements in the other plasmid, remove the bit of DNA I had prepared along with the marker gene and the white gene, and put it inside the DNA of one of the cells in the egg that would become a germ cell. The germ cells are the cells inside an embryo that will become the sperm or eggs of the adult.
If it worked, that means that one, maybe two, germ cells would carry my DNA, plus white, plus lacZ. The egg, if it survived, would produce a adult fly, one or two of whose sperm or eggs would carry the addition. The flies born from those sperm or eggs would have red eyes, because of the functioning copy of white they got with the rest of my prepared DNA. It takes about two weeks for the eggs I inject (the ones that survive) to become adults, and another two weeks for their children to mature.
I must have injected over a thousand eggs, and yesterday I got my first two transgenic Drosophila. Lovely, lovely, dark red eyes with orangy patches which are apparently characteristic of such transgenic flies. It was just in time to go into my second-year report as well. I'm so happy ^.^
- Sun Kitten, 22nd July '03