Merlin is on track

Work marches on slowly. I did a PCR of wild type and a mutant (serine to alanine at position 518) Merlin. I actually got a band on the gel at the correct place but was still not sure if I successfully added the correct restriction enzyme sites to the 5' and 3' ends. So, I sent the PCR products in for sequencing and lo and behold, 8 of the 10 sequences were correct. Cause for a minor happy dance. Next, I will do a DNA clean up reaction. This will essentially filter out all the extra stuff from the PCR reaction such as residual enzyme and salts, leaving me with pure DNA. I can then proceed to do a restriction digest of both my PCR products (to "rough up" the ends) and of the viral vector that I will be dropping the PCR products into.

I just hired a student to work for me for the summer. He has no lab experience but is working out well. He is smart and is a hard worker. He has already gotten some results this week on a project that my last student (who just graduated and is going on to dental school) started. It is a pretty simple project but has been giving us headaches. One of the limitations of our work is the availablity of tumor samples. Many labs get around that problem by using cell lines. Cell lines are great in that they can be propagated for years and years (HeLa cells are cervical cancer cells that have been around since the 1950s) and are available whenever they are needed. On the flip side, immortalized cell lines are also a screwed up. You have essentially taken what in most cases are normal cells and made them into cancer cells so that they will continue to grow. This means that your results need to presented with the caveat that what you are reporting may not necessarily be exactly what happens in vivo. We all accept that and continue on with our work. We would like to be able to work with a cell line because we wouldn't have to be so dependant on someone getting cancer. However, there are no good vestibular schwannoma cell lines. As such we decided to make our own. We have two cell lines that we are working with and as part of the characterization process we are attempting to sequence the gene that encodes Merlin. We have isolated genomic DNA and are in the process of PCRing managable parts of the gene for sequencing. We have been having problems finding the right conditions but yesterday we actually got it right. With one part down, we have about 10 more to go. Let's keep our fingers crossed.

Old McDonald I am not - it just seems that way

I think Dad always wanted one of us to become a farmer and follow in his footsteps. It would appear that he has been granted his wish - kind of. Instead of raising hogs, I am going to be raising rats. We normally order in one pregnant rat every week but they are expensive. We were ordering from a company called Harlan but they were charging us just over $100/rat. I found another vendor that carried the same type of rat for about a third of the price. I was pretty happy about that until I noticed that the number of pups per litter was severely reduced. There also seems to be quite a bit of cannabalism going on. I know that happens with hogs occasionally also. I think we are being sold first time mothers who just aren't having very big littters (less than 8 pups). As such, we are going to start our own breeding colony. This will save us a lot of money and hopefully provide us with all the pups we need. Rats tend to be quite prolific. Their gestation period is only 21 days and their estrus period is only 4 days.

If it worked the first time, we would just call it search

I have been unsuccessful finding a clone that contains any one of my merlin constructs. It's a good thing that there is a plate full of bacterial colonies to pick through. I only need one colony to of each construct.

Today I am switching gears a bit and doing some cell culture. I have six plates of vestibular schwannoma cells that I am going to treat with a kinase inhibitor. I will let them incubate for a couple of hours, isolate the protein and run Western blots. Pretty straightforward and simple. I also have a couple of dissections to do this afternoon. I am going to remove the cochlea from a couple of mice. One of them is what is called a knockout mouse and the other is wild type. The knockout mouse has a gene, in this case a neurotrophin receptor, "knocked out" of it's genome. Additionally, one ear on each mouse has been deafened. I will begin by perfusion fixing the brain with paraformalydehyde. Then I will remove the temporal bone and proceed to dissect out the cochlea. After removing the stapes from the cochlea, I will make sure that both the round and oval windows are open and finally place the cochlea in fixative. Once fixed, I will de-calcify the bone with EDTA (yes, the same EDTA in the ingredient list on your box of Fruit Loops). Following de-calcification, we can section the entire cochlea into 7-10 µm thick slices, place them on a microscope slide, and label them with fluroescent tags to various proteins. By labeling the appropriate proteins, we can begin to get a sense of what role our neurotrophin receptor plays in deafness. I'll keep you updated at to our results.

Back to Merlin

I am finally getting back to some molecular biology. Although I don't particularly enjoy working with DNA, it is vital to what we do. Last night I inoculated some media with a colony of bacteria that I am hoping contains my plasmid. When I came in this morning, I noticed that somebody had turned the incubator down to 30 degree C. I should have checked it when I put my cultures in but in reality, the incubator has never been at any other temperature but the 37 degrees C necessary for optimal bacterial growth (at least for the vast majority of bacteria used in molecular biology). My cultures still grew but not as well as they should have. I turned the incubator back up to 37 and am letting the cultures grow for a couple hours more. After they are done, I will isolate the plasmid DNA and do a restriction digest to see if my desired DNA sequence is actually there.

Hah!

Well, I redid the experiment and got cleaner results. At first I was going to mess around with the amount of protein I loaded but as I thought about it, I got the distinct impression that the only thing I should change was the concentration of blocking buffer - which is something I never do. Blocking buffer is usually a milk or albumin solution that helps to reduce non-specific antibody binding on the Western blot. I have always used a 5% solution. Yesterday, as I was planning the experiment, I got the distinct impression to use a 10% solution. I did and my results today are easily understandable and interesting. I found that pERK signalling is increased in vestibular schwannomas while not in control tissue. Although we expected this, the results are gratifying none the less. However, I didn't see any differences between the two tissues with regard to pAKT signalling. We didn't expect this. So what does this mean? At first glance, it simply means that growth of schwannomas is regulated through the ras-raf-MEK-ERK signalling pathway and not so much through PI3 Kinase. So why is this important? Well, the more specific a response is within a cell, the easier it is to control abnormal behavior. Perhaps someday this knowledge will lead to more effective treatment options for patients with this type of tumor.

ARRGGG!!!

I am a little perplexed right now. I have been doing protein work for 10 years now and usually when something is what I expect I can at least come up with a reasonable explaination and then a follow-up experiment to test my hypothesis. My results from last week are so contradictory that I am just running the experiment over again - same conditions. If I get the same results I will at least know that the odd results are not due to my technique but are due to tissue variability. I just want to get this set of experiments done and move on to the next thing. I think all researchers are a little ADD. Routine doesn't sit well with most of us. That is the primary reason I left my last job. I was doing the same thing day in day out. After a year and a half of that, I was going nuts. Here's hoping for good results.