![\"\"](\"http:\/\/www.lablit.com\/images\/Cloning.jpg\")
\nStrange fruit<\/strong> Not so much a Blind Watchman as a gardener with 20\/20<\/em><\/p>\n
\nAs with any endeavor, there are tasks in science that I enjoy and tasks that I don’t; there is also a strong correlation, for me, between enjoying a task and being good at it. I happen to be a whiz at making stable cell lines: coaxing bits of foreign DNA into immortalized cancer cell lines and selecting out a new strain derived from a single mutant cell. In this procedure, which is more like horticulture than molecular biology, I can induce the evolution of practically any trait that is not lethal to a cell by using a particularly nasty form of artificial selection and letting nature take its red-toothed and clawed course.<\/p>\n
\nA few days before my ski holiday, I transfected two different DNA constructs into a single plate of cancer cells, one encoding a histone protein fused to a fluorescent red tag and the other encoding a newly published actin-binding domain I was eager to try out, fused with a green fluorescent tag. The first construct also encoded a gene conferring resistance to the deadly drug puromycin, while the second construct would allow cells to escape the effects of the equally iniquitous chemical G418. One the second day, I diluted the transfected cells across a series of plates so that the modified clones would not be too crowded to isolate once they began to grow. While I was off on the slopes, one of my lovely colleagues obligingly nuked my Petri dish with several doses of the drugs and watched while hundreds of thousands of cells died off, filling the medium with light-refractive clumps of unhappy corpses. When I got back, the carpet-like monolayer of cells I’d left behind had almost completely vanished, but when I held the plates up to the light and examined their undersides, I could see the faintly glowing, pale spots of resistant colonies scattered across the plastic.<\/p>\n
\nIsolation is satisfying: you wash off the medium, pluck up a sterile glass cloning cylinder with a forceps, dip one end in high vacuum grease and press it down around one of the colonies to form a water-tight seal. A tiny amount of the enzyme trypsin will loosen the colony (consisting of anywhere from twenty to a thousand cells) and the cells can be replated into a small-welled vessel to begin the laborious process of nurturing, expansion and validation. I usually take about twelve colonies to ensure that I get at least one cell line that is healthy and that expresses both markers to sufficient levels, but for the next week or so I’ll have my hands full transferring my new babies to increasingly larger vessels, freezing down samples as back-ups and running tests to decide which will be ultimately be chosen — and which will be flushed. <\/p>\n
\nIt’s a jungle out there.<\/p>\n","protected":false},"excerpt":{"rendered":"
My loyal readers may have noticed that Mind the Gap was silent on the topic of Darwin this year. I have nothing against the man, and admire his work as much as the next girl. I suspect, however, that there … Continue reading