Sydney's bushfires have been temporarily muted by almost a day and a half of blessed rain, and as I walked to work this morning I was continuously aware of the strange absence of the smell of woodsmoke in my nostrils. The smoke has been part of our lives for several weeks, sometimes distant and sometimes all too close, just as it is nearly every summer. To walk through a world of clear blue sky and clean air seems almost unreal.
The lab is hectic, with everyone struggling both to make up for time missed over the last week (many of us were either protecting our homes from the fires or unable to get to work due to fire-related roadblocks) and to wrap experiments up before Christmas. Conversation is stilted, unusually for a lab that is notorious for its boisterous atmosphere. This is fine by me, and I make the most of the lack of distractions.
My major aim today was to complete the generation of my knockout construct, a piece of DNA which will be used to delete a specific gene in a breed of mouse. The effect on the mouse of deleting this gene will give us information about the gene's function, adding to the information my lab has already gathered on its role in humans. Due to unforeseen complications, making this piece of DNA has taken me much longer than anticipated - almost an entire year - but the final steps in its generation have been comparatively straightforward. Two weeks ago I introduced the completed construct into bacterial cells via the rather Frankensteinian technique of mixing DNA and bacteria together and then subjecting the cells to a brief but powerful pulse of electricity. I then selected cells which contained a copy of the construct and allowed them to grow in very large numbers. Each bacterial cell in the resulting bacterial culture contained a faithfully replicated copy of my construct, which I then extracted by dissolving the cells using a combination of detergent and osmotic pressure. Finally, I purified the DNA from the rest of the bacterial debris using salt and ethanol, leaving me with a small volume of solution containing a very high concentration of my desired DNA molecule.
My task today was to take this molecule, which was present in a circular form (allowing it to be replicated by bacteria), and convert it into a linear molecule. To do this, I needed to cut the molecule at a single site (imagine taking a rubber band and cutting it in one place, turning it into a rubber string). I did this by using a commercially available enzyme which cuts DNA molecules at a highly specific sequence, CCGCGG, which is present at only one site in my construct. After a year of toil and heartbreak, the generation of my construct was completed in one pathetically easy step: the addition of DNA, enzyme and a few chemicals to a plastic tube, which was then incubated at 37 degrees Celsius for two hours. And then - hey presto! - a linearised construct.
Now there is just the final, tedious task of confirming that my construct has indeed been cleaved by the enzyme, which I am doing as I write this daylog. I took a small sample (only five microlitres) of the completed reaction and loaded it onto an agarose gel, then applied an electric current to it. The DNA, being negatively charged, will migrate through the gel towards the positive electrode, and the speed at which it migrates will differ depending on whether it is circular or linear. By staining the gel with a chemical which emits visible light when exposed to ultraviolet radiation I will be able to identify how far my DNA has migrated, and thus whether all (or even some) of it has been successfully linearised.
And what's the point of all this? Well, the next step in my project will be to take my linearised, purified construct and introduce it into mouse embryonic stem cells using the same technique I used to get it into bacterial cells (electrocution). I have designed my construct so that it binds specifically to a particular region of the genome of these cells - the gene I want to delete. In a small fraction of the embryonic stem cells, the construct will bind to this region and undergo a process known as homologous recombination, which will delete the target gene and replace it with another gene which makes the cells resistant to a toxic drug. When the cells are grown in the presence of this drug, only those which have undergone the required gene deltion (and thus contain the resistance gene) will survive. I will take these cells and inject them into early mouse embryos, and then implant the embryos into mouse foster mothers where they will develop as normal, but will contain a few mutant cells derived from my embryonic stem cells. If I can find a few mice where these cells have been incorporated into their germ cells (sperm or eggs), then I can breed them to produce mice who have the mutation in every cell in their body.
But that's all in the future. This afternoon I will focus on confirming that the construct has been successfully cut, and if it has I will then purify it and prepare it for its introduction into embryonic stem cells on Friday next week.
Beyond the sterile, uncluttered benches, outside the sturdy lab windows, the sky is still clear blue and free of both smoke and clouds. In a few minutes my beeper will sound, indicating that my gel is ready for inspection. The repeated failures and disappointments of this year have left me cynical about even the simplest experiment, but today - perhaps due to the weather, the temporary relief from the bushfires, the proximity to my Christmas break or a combination of all three - I feel curiously optimistic about success. I guess it's time to go and see if that optimism is justified.