Diary of a Cystic Fibrosis Researcher Page 1 |
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Kept by a Graduate Student in: Teintze Research Group: Dept. of Chemistry/Biochemistry
Monday 9/25/00 Finally got what looks like protein (CFTR) on my western blot (right). It's kind of in the right place near the top of the gel, but as usual, the whole 2 day procedure needs to be repeated. Research is full of inconclusive results I'm finding out. I sometimes think the only two things I've gotten good at in research are running western blots and breaking large beakers. Some background: My lab has been trying since the beginning of last Summer to put the gene for CFTR in a baculovirus (so I can then use this virus, which infects only insect cells, to grow up the CFTR protein in the insect cells. If we had to use human tissue to provide the CFTR protein the whole thing would be impossible due to the limited availability of human tissue, of course. While we already have the CFTR gene in another baculovirus and this works just fine for us, the new one we are currently trying to "make" will have a poly-Histidine tag on the end which will make it easier to purify, which is a whole nother story). For more background click here Tuesday 9/26/00 Talked to my PI today (principle investigator or boss) and he recommended going forward with the next step, which is to do a "plaque assay" even though the results from yesterday are still inconclusive. He believes that since the "plaque assay" and the western blot are both about the same amount of work (and time!) and we will get the same answer with either technique, we might as well proceed as if we have the right virus with the right gene (i.e. CFTR gene) in it. Works for me.
Wednesday 9/27/00 I'm still waiting for the insect cells to come up (i.e. grow and divide) in order to do the plaque assay. I need probably about 20 million, which is about 3 good-sized flasks worth (one flask is about the area of a person's hand). Cell culturing can be tedious. The first time I tried it (my first summer in the lab in 1996), I had to start over 5 times. Either the media (food for the cells) kept getting contaminated with bacteria or fungi or the cells would simply die for reasons known only to themselves. Since 1996, I've gotten more careful, but as far as I'm concerned cell culturing is still more of an art than a science. I looked at my western blot results again from Monday (pictured on top, right). The membrane dried out and the background bleached out enough since then so that a definite band running exactly the way CFTR usually does on a gel is clearly visible. It's definitely CFTR protein (the good news), unfortunately, not a lot (the bad news). In fact, there's so little CFTR on the membrane that I have to be in exactly the right lighting (i.e. near a window) to see it at all. Probably a total of about 50 billionths of a gram, which is much less than a single grain of salt weighs. But the main thing is that he and I saw the protein on the membrane, which is encouraging. This means that the cDNA (gene) for CFTR is in the baculovirus and probably doesn't have any serious mutations in it, and will give us active protein in the near future. (editor's note: at this time back in September, we believed we had been working with the correct, non-mutated CFTR gene. This assumption would later come back to haunt us. Read on.....) Friday 9/29/00 The insect cells finally grew to the point where I think I can try a "plaque assay". Plaque assay is where one tries to isolate the virus that has their gene in it from all the other viruses that don't. The ones that don't have the gene, if left to their own devices, would eventually overtake the good viruses with the CFTR gene in them; the end result being that that you loose the virus with the gene that will make your protein. The first time I tried this procedure, which involves pouring hot agarose directly on top of a single layer of insect cells (agarose is a kind of hot jelly that quickly solidifies after you pour it) all the cells died. Not a pleasant thing to see first thing in the morning. I poured hot agarose onto 6 plates today (at 5:30 p.m.). It was tedious, as usual. I have to be sure and do everything in a sterile manner, using what is called "aseptic technique", wearing gloves and placing my hands inside a biosafety hood, which is a metallic and glass station in the lab about the size of a volkswagan. It makes an annoying buzzing sound each time you have the window (thru which your arms are going into) at the wrong height. I will cross my fingers and wait until tomorrow morning. More waiting..... Saturday 9/30/00 (9:00 a.m. MDST) Good news finally! Nearly all of the insect cells survived the hot agarose treatment. This is the first time all of them have made it thru this procedure (which can only be a good sign). It's still too early to know whether it worked, but so far so good. Culturing insect cells can be a daunting business even when you don't do anything harsh to them. For them to survive hot agarose is a breakthrough for the lab. (if they survive a few more days, that is). The next step is to wait 3-4 more days (yes, more waiting) and hope the agarose starts to turn blue in spots on the petri dishes, which is a sign we have the right virus. For more on how and why we use the color change to detect for "good" virus, click here... |
Western Blot (picture taken: 9/25/00)
*Purple band at top of membrane on right is, I'm hoping, CFTR. The other purple lines are probably artifacts from the gel.
* Petri dish with a single layer of insect cells on bottom. Hot agarose is being poured on top because, once hardened, it keeps the virus from moving around so we can better isolate the one we
will use later (the virus with the CFTR gene in it). There is also a chemical in the agarose that turns blue if the right virus is being produced so we can see it better.
*Insect cells (which came from the ovary of a butterfly back in the 1970s). The arrow is pointing to virus which has infected them: the same kind of virus we are trying to produce. This is what I see when I look at the cells in our lab under a microscope.
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