Cre-lox

The IP's I spoke about in my last post are still ongoing. I have had some success with them but the results are too ambiguous to make any real conclusions yet. However, in our ongoing attempt to understand how Merlin is involved in cell signaling, we have obtained an new strain of mouse. Actually two new strains of mice. These mice utilize the cre-lox system to knock out my gene of interest. I'll try to describe it in general terms. First, one mouse is genetically modified so that my gene of interest (nf2 or merlin) is bordered on both sides by a sequence of DNA called loxP. A second mouse is engineered to contain the DNA for an enzyme called Cre recombinase. When Cre recombinase finds a loxP site, it snips it and recombines the remaining DNA (see below).

In our case, we are interested in making a deletion. Now the coolest thing about the particular Cre mouse we have is that we can control where and when the enzyme is expressed. The where is controlled by a sequence of DNA found upstream of the enzyme called a promoter. In this case, the promoter is specific for schwann cells. The when is controlled by an estrogen receptor that is sensitive to tamoxifen. When I breed a cre mouse (a hemizygote) with a mouse containing loxP sites (a homozygote), I get offspring that are able to knockout Merlin expression when I treat them with tamoxifen. How cool is that?

I just got my first round of mice treated and performed my first Merlin IP in order to determine if the system worked. I have yet to run the gel but when I do, I will post the gel.

Science rules.

IPs

I almost titled this post Looooong month but decided I need to quit complaining. I think most of our new people have settled in and feel somewhat comfortable. I am still worried about our new Korean research fellow. He is certainly bright enough to do the work (an MD PhD) but he either doesn't understand me well enough or he just isn't listening. We'll get through this.

Lately I have been doing some experiments with some mice that have been bred to have a mutated NF2 (Merlin) gene. Merlin is the tumor suppressor protein on which we focus the majority of our efforts. We know that when this protein is missing or when certain areas of the protein are mutated, schwannomas, menigiomas, and several other 'omas develop. So what I have been doing is harvesting sciatic nerves and isolating protein. Following the isolation protocol, I am probing the tissue lysate for two receptors that are potentially upregulated in cells without properly functioning Merlin. One of the things that we know based on earlier evidence from our lab (and others) is that one of these receptors, ErbB2, is localized to an area of the cell membrane that is enriched in cholesterol and other lipids. These areas are called lipid rafts and have been shown to be rich in certain types of receptors. The problem with lipid rafts is that they are very difficult to solubilize without the use of harsh surfactants. I have prepared two lysate fractions, a Triton X-100 (a light surfactant) soluble fraction and an SDS (a harsh surfactant, also called Lauryl Sulfate - see your shampoo bottle) soluble fraction. I have run them both out on a polyacrylamide gel, transferred the proteins to nitrocellulose and finally probed the membrane for my receptors with antibodies. Unfortunately, I have been largely unsuccessful to this point. Today I am finishing up an immunoprecipitation of ErbB2 in an effort to enrich the sample I am loading onto my gels. It is a fairly straight forward proceedure. I simply incubated my lysates with the ErbB2 antibody overnight in the cold room and then incubated my antibody:antigen complexes with very small beads that bind to the complex. After washing off any contaminates, I will elute ErbB2 by boiling the beads in a buffer and collecting the buffer. Hopefully, this final buffer will be enriched in ErbB2. I will run the gel tonight and do the Western blot tomorrow. If this works, we will be on our way to getting this project off the ground.

Looong week!

The first week of school usually just means that there are more people walking the halls for me. However, the first week of school this year coincided with five new people starting in the lab. We have a new Korean research fellow who will be with us for a year, a surgical resident doing a two year research rotation, two medical students (only 4 weeks each) and an undergrad who is doing a research project for an Integrative Physiology class. Oh, and I also hired another undergrad for a workstudy position in the lab. All of them are very nice and quite intelligent but have very limited experience in the lab. I have literally had people lined up to ask me questions. I am tired and would like a few minutes to do my own work. It doesn't look like that will be happening any time soon but it never hurts to dream.

One of the good thing about having new people in the lab is that we can start new projects. Perhaps I am just a glutton for punishment but I really enjoy the excitement of a new project even though it means more work (which is a lot better than having no work). I like being able to explore new ideas with no real idea what the data are going to show.

As new projects start, old ones end. Today was the last day of treatment for my tumor-implanted mice. I will be doing post-treatment MRIs next week and then sacrificing the animals and removing the tumors for sectioning and immunolabeling. I'll post pictures as they become available.

Cool picture

Here is a picture of a mixed population of dissociated cells from the spiral ganglion neuron. Schwann cells are green (probed with an antibody against S100) and neurons are red (probed for NF200). The blue color shows the nuclei of all cells regardless of cell type. As you might imagine, trying to get a pure population of any of these cells types is difficult at best. Luckily there are very few times that I need to do that.
These cells are being grown on an unpatterned bed of polymethacrylate. This experiment was essentially a proof of principle experiment showing that cells will attach and grow on polymethacrylate. I have another batch growing on patterned polymer as I write. They should be ready for processing by tomorrow.

Cell culture and cochlear dissections

While I wait for my boss to get done seeing a couple of patients so that we can have lab meeting this morning, I thought I would write a couple lines (more likely a couple of paragraphs).

With Ningyong gone, I have been doing a lot of dissections. Last week, I dissected the spiral ganglion out of the cochlea of about 9 rat pups. After dissociating the ganglion into its individual neurons (there are about 30,000 neurons per spiral ganglion), I cultured them on some specialized substrates. We are doing a study with a researcher in Chemical Engineering who uses different polymethacrylates (contact lens are a type of methacrylate) to create patterns. We are having him create some slides for us with what are essentially furrows of differing widths. We are trying to see if we can direct the axons to grow along the furrows. We had some intial success but the last batch of slides I got were in pretty rough condition. I don't know if I will get any useful information from these polymers. When I get things worked out, I will post some pictures. The first round of images were pretty impressive. Eventually, this study could lead to a method of improving the interaction of neurons to cochlear implant electrodes thereby improving the level of hearing in cochlear implant patients.

Just pile it on!!

One of the rocks of the foundation of our lab has crumbled. Ningyong has moved. She has been working in our lab for almost 7 years. Her husband just accepted his first faculty position at the University of Southern Alabama so they have moved down to Mobile. Ningyong was a true workhorse. She did everything she was asked to do without complaint and did it well. I am going to miss her. Her departure was a bit sudden so we didn't have time to find a replacement for her before she left and since the hiring process at the UI takes nearly 3 months, I am going to have double the amount of work to do for a while. This means that in addition to the every day administrative stuff I normally have to do and my own research, I now also have two animal colonies to care for (one rat colony and one mouse colony) and three additional projects to work on. At least I have a job! I just hope we can somehow speed up the hiring process and get work back to normal.

Am I really this dumb?

So I got my minigenes in the last week and proceeded to go through the cloning proceedure once again - no luck. I knew it was going to be a difficult experiment. I am attempting to insert a small sequence (only 51 base pairs) into a large (7900 base pairs) plasmid. This sequence is called a nuclear export sequence. Essentially it is like a zip code. It directs the gene product (a protein) to stay outside the nucleus. We are inserting it upstream of a gene I have already cloned into a plasmid called p75. P75 is a neurotrophin receptor that plays several different roles depending which neurotrophin it binds to. The dogma is that p75 is a death receptor. Upon binding to the proper ligand, the intracellular portion of the receptor is cleaved and transported to the nucleus where it causes the cell to undergo apoptosis (programmed cell death). We want to prevent the intracellular portion from entering the nucleus by attaching this nuclear export signal to the protein. Back to cloning. I was sitting in the temple the other day for a wedding and the thought occured to me that I should attempt to clone p75 into the plasmid containing my NES rather than the other way around. That way I would not have the difficulty associated with trying to drop a very small insert into a large plasmid. What a great idea. So the next day I hustled back into the lab and anxiously pulled up the computer files with my sequences to determine the best method to do the experiment. While examining the sequences I realized that I had made a fundamental mistake when designing the NES. It was such a colossal mistake that even if I had been successful cloning the NES onto the p75 gene, it would have been non-functional. When I ordered the NES, I switched the restriction enzyme sites. Restriction enzyme sites are small (usually 4-6 bp) sequences of DNA that are specific to certain enzymes that will cleave the DNA at those positions. When I cloned p75, I had used PCR to insert a couple of enzyme sites in a specific sequence (EcoRI then SpeI) for the eventual addition of the NES. However, when I ordered the NES, I did so with SpeI first then the NES sequence followed by EcoRI. This means that had I been successful cloning the NES into the p75 plasmid, the NES would have been inserted backward, rendering it unreadable. What a stupid mistake! I am glad I found it. Had it not been for that flash of inspiration in the temple, I would never have realized it until I sequenced the final product. I am really irritated that I have spent so much time, energy, and money on something that wouldn't have worked anyway. I reordered the NES on Thursday and hopefully will be successful with this project soon.

On a happier note, I have a job for at least another five years. We just received word that we were awarded an RO1 grant from the NIH. RO1's are the lifeblood of the biomedical sciences. Faculty members lose their jobs if they fail to get grants. We are very fortunate to have gotten this grant and are breathing much easier. We still had a couple years of funding through other grants but this grant is the big daddy. It will provide us the resources to really start growing and doing better research.

Cloning frustration

Sometimes the brilliance of my own stupidity explodes like a star over my head. I have been working on a cloning project off and on for a couple of months now in between other projects. About two weeks ago, I decided that I needed to get it done so that we could move on and finish the experiments that we had planned for these genes. The dumb cloning procedure is relatively easy but I just couldn't get anything to work. Then, a couple of days ago, I realized that the insert I am trying to add was single stranded DNA instead of the necessary double stranded DNA. I had neglected to order the complementary strand of DNA and therefore hadn't annealed the two strands together. What a stupid mistake. You'd think I was an undergrad doing this for my first time. I reordered my sequences of interest (a nuclear export sequence and a nuclear localization sequence) yesterday as double stranded DNA. Hopefully this experiement will work now.

I am also in the middle of treated my mice that we implanted. One of them has died but since the daily injections started by everybody else seems healthy. Only 10 more weeks to go.

More stuff

I just got done editing 11 pages of a manuscript we are writing. My brain hurts. This paper will describe all of our JNK experiments as well as a few others to complete the story. I think it is a pretty strong paper and we are going to send it to the Journal of Biological Chemistry. JBC is one of the big journals in the biomedical sciences so we really have our fingers crossed.

The initial MRIs were completed yesterday and the real work of treating the 24 mice on a daily basis for 3 months will begin shortly. This will be one of those things that I will just have to grin and bear. It isn't particularly difficult (I've done this once before with far fewer mice) but it is time consuming.

Rant for the day

Perhaps the bane of my existence in the lab is bureaucracy. It comes in many forms but primarily it exists in the form of the the Animal Care Office and the Health Protection Office. Both offices perform an important function but for heaven's sake, could there be any more hoops to jump through. For any experiment we perform involving animals, we must have an approved protocol that covers everything from my anesthesia method to the type of suture material I am using to the frequency of post operative observation. Now don't get me wrong, I am not against oversight. Actually, I believe oversight of animal care is vitally important. However, it can get a little ridiculous. Research is an evolving process which means that I have to continually amend our animal care protocols as things change. Paper work is a pain.

The Health Protection Office is even worse. They are continually changing the rules regarding chemical storage and so forth. Don't store acids and bases together, chemicals can't be on a shelf without a lip, certain chemicals must be in secondary containers, and so forth. There are also a number of online tests that we must take each year. I am supposed to ensure that everyone in my lab is up to date but they won't give me access to their database so I am unable to see who needs to take which test.

The saving grace in all of this is that the people who come in to audit all the labs are easy to work with and easy to get along with.

For your viewing pleasure

How cool is this?

This is a longitudinal section of a live mouse (T2 weighted MRI scan). The tumor is just under the skull, at midbrain.

I have got the coolest job.

Success is sweet

I saw tumors. The MRIs were successful. It always amazes me to see so much detail in an MRI. The technology involved is really quite straight forward (it has been used by chemists for decades) but the application of the technology is really ingenious. Too cool.

MRI tomorrow

The JNK experiments are finally done - at least for now. I am getting ready to do some in vivo experiments. I think I described the experiments awhile ago but they failed when we did them in December. Briefly, I have implanted human tumors into mice that have been genetically bred not to reject foreign tissue. The first time I did this experiment I simply made a small pocket under the skin between the shoulder blades, inserted a chunk of tumor, and sutured it back up. This time, I not only placed tumor between the shoulder blades, but I also drilled through the cranium, exposed the brain, and placed a small piece of tumor inside the cranial vault. Since the tumors we deal with are found in the brain, this approach more closely approximates what is actually going on in a human patient. I had 15 successful surgeries. Tomorrow, I am going to take 5 of the mice and give them MRIs to establish a baseline tumor size. Once all 15 mice are scanned, I will begin a daily 12 week treatment regime that will culminate in a final MRI (to establish an endpoint tumor size) and a removal of the tumor for immunocytochemical analysis.

I am also thick in the middle of some cloning experiements. I am trying to clone a nuclear export signal or a nuclear localization signal onto the front of the p75 receptor gene. Once this is done, we will be able to transfect cells with these molecular constructs to get a better idea of what p75 does in certain environments. Wish me luck.

JNK

I have been devoting almost all of my time to JNK (pronounced JUNK) lately. In my last post I mentioned that I had stumbled upon something interesting. The premise of the experiment was that we would compare the levels of JNK activity in cancerous tissue to non-cancerous tissue. Not too big a deal. JNK is a multi-tasking protein. Actually there are three JNK proteins. The ones I am concerned with are JNK 1 and JNK 2. They are very similar in sequence and are usually lumped together. The phospho-antibody I have to look at activating JNK is actually designed for both JNK 1 and 2 but I have separate antibodies for the inactive forms. JNK can be involved in cell suicide (apoptosis) or cell survival depending on the stimulus and cell type. We hypothesized that JNK activity would be increased in our tumor samples. That doesn't appear to be the case at first blush. I was a little disappointed at the results but it is what it is and I accept that. The interesting part came when I stripped the antibodies off the Western blot and re-probed for the inactive versions. What I found was that JNK 1 and 2 are being differentially regulated in these tissues. Attempting to find out why that might be, I searched the published literature and found a precedent for this in a paper published 5 years ago. We are working in completely different cells but it will be interesting to see if this plays out. The published paper showed that cells lacking JNK 2 proliferated more rapidly. Our tumors appear to have reduced JNK 2 expression. This is cool.

I am going back and re-running the Westerns, this time probing for the JNKs independantly. The JNK 1 blot I finished today was not very convincing. We will see what the JNK 2 says tomorrow. This could lead to a bunch on new experiements.

I have been chiding myself for not posting hear (get it?) more frequently. I am aware that 99% of anybody reading this blog probably doesn't really understand or perhaps even care (and that doesn't bother me; I am writing this more for me than for anyone else) what I am talking about and that is probably why I have been slacking. Another reason is that Biomedical research crawls forward at a glacial pace. It is rare that something new and ground breaking occurs. I figure that if I can have even a couple of those types of events occur in my career Iwill be pretty happy. Mostly, though, it is millimeter by millimeter that progress is made.

I mentioned the xenographs I was going to do in my last post. We finally performed the surgeries in December but with disasterous results. Out of the 16 mice we implanted, only three of them survived. I am not really sure why but I am guessing that it is a combination of anesthesia problems and internal bleeds. There are a couple of large arteries near the implantation site that I know in at least a couple of the mice were nicked. Placing the tumor next to one of those arteries within the cranial vault may also have caused the mouse to stroke out due to increased pressure on the artery. The human patient who so kindly let us have his tumor will be back in surgery in a few months so we will give it another try.

I just finished a Western blot that is quite interesting. I need to spend some time thinking about it before trying to describe the results. If what I think is occuring actually is occuring, I may have stumbled onto one of those moments - a small one but one never the less. One of my favorite things about biology is that there is always something new to think about and figure out.