ChIP

Finally. I finally have some preliminary results. After 3 months of chasing this down, I have 190 µl of DNA at a concentration of about 15 ng/µl. Whew! Now the hard work of going from an easy to grow cell line to finicky primary cells that we only get about once every two weeks or so. There are so many ways this can go wrong. The experiment takes about three days to go from living cells to purified DNA in a tube. One poorly aimed sneeze and I have to start over. Sometimes I am amazed that science ever moves forward at all.

What worries me about this experiment is that I will be successful in pulling down DNA that is bound to p75 but we won't be able to align it to the human genome. In other words, I will have done a lot of work for no results. A part of this worry stems from the fact that this is essentially an in Vivo test tube experiment. The cells I am currently using bear little resemblence to the real thing. Once a cell has been immortalized, it's genome is frequently messed up. This could mean that any results we obtain do not accurately reflect the interactions in a normal cell. We will be getting around this by using cells that we harvest directly from human patients. However, that only solves one issue. The other issue is the p75 DNA construct I am using. I have modified the original gene by cleaving off the part that normally is in the extracellular space (the ligand binding domain) and tagged it with two different DNA sequences. One tag is for identification purposes and the other is the NLS tag which forces p75 to translocate into the nucleus. This is a rather contrived system. We know that in a normal tumor cell (and I use that term loosely), p75 is a single pass, transmembrane receptor. Once it binds it's ligand, the intracellular part is cleaved and is translocated into the nucleus. My construct skips the ligand binding and normal intracellular trafficking. However, what if the normal trafficking mechanism is necessary for proper DNA binding? What if p75 is associating with another protein as it is being shuttled into the nucleus and it is that protein that is binding to the DNA and p75 is simply acting as a co-factor? Maybe p75 is not associating with another protein but what if it is being modified in some way while in the translocation process thereby allowing it to bind DNA with greater fidelity or greater promiscuity? If the translocation process is important in p75-DNA binding dynamics, I won't know that with my construct.

These are the things I think about as I am trying to fall asleep at night. At least they are entertaining and intellectually provoking.

ChIP update

Science moves at a snail's pace most times. I finally have the first part of this experiment nailed down (DNA isolation and cleavage). I was using a sonicator to break up the DNA but was getting poor results. I went to an enzymatic method called Micrococcal Nuclease digestion and have had some success. I am going to run through this once more tomorrow and attempt to clean the results up by combining the MNase digestion with sonication. Next week I will move on to infecting the cells with virus so that p75 will be expressed and then work on getting p75 binding to beads for the immunoprecipitation part of the experiment. Once I have that worked out, I will change cell types and make whatever small changes I need to make before really hitting this experiment for results.

New experiment

I am still working on the experiment I wrote about in the last post but I am now adding on another experiment that takes the next step. The first experiment (from the last post) will confirm that my protein of interest, or rather a portion of my protein of interest, is exerting intranuclear control. The protein I am looking at is called p75 which is a single pass transmembrane receptor. This means that a portion of the protein sticks out into the environment, a small portion passes through the cell membrane and the remaining portion (the intracellular domain or ICD) sticks into the cell. Now, unlike fruit fly scientists who, in attempt to be funny, name their genes things like sonic hedgehog or SOS (son of sevenless), most of the rest of us are pretty boring. P75 is protein (the P) that has a mass of 75 kilodaltons. P75 is a receptor protein that functions to bind neurotrophins such as Nerve Growth Factor (NGF). NGF is call a ligand. The whole purpose of a ligand binding to a receptor is to cause a change in gene expression so that a cell can respond to it's surroundings. This process of intracellular communication or cell signaling is what fascinates me. This process is very dynamic and can be relatively simple or incredibly complex. The process often depends on the environment and the context in which the signal is received by the cell. For example, the accepted dogma for p75 signaling is that when p75 binds to a ligand, an enzyme called gamma secretase clips off the ICD which then through some mechanism is transported to the nucleus where it causes the cell to undergo apoptosis (programmed cell death or more literally, cell suicide). However, this is not always the correct. We have shown that our tumor cells actually proliferate under treatment with certain p75 ligands. Also, recent research from another lab in NY has shown that the p75ICD binds to the promoter region of a gene called Cyclin E1. Cyclin E1 is the protein that jump starts the synthesis of DNA replication in a cell in order for that cell to undergo mitosis. In light of these data, we have decided to see to what else the p75ICD is binding once it translocates to the nucleus. We are doing this by a method called ChIP Seq. That stands for Chromatin Immunoprecipitation sequencing.

In short (the protocol I am using is 15 pages long, so yes, this will be short), I will be forcing p75 to go into the cell nucleus where it will presumably bind to whatever sequence of DNA it binds to. I will then crosslink p75ICD to the DNA with formaldehyde, do a nuclear isolation and then clip the DNA into lengths of about 200-500 base pairs with a sonicator. Once I have confirmed the length and amount of DNA with it's associated proteins, I will apply the DNA/protein to some magnetic beads that are coated with an antibody that recognizes only p75. Once p75 and it's associated DNA is bound, I will isolate the beads, wash away any extra, non-bound protein/DNA, finally elute the p75/DNA into a small tube. Once I have done this, I will reverse the crosslinking, degrade any residual protein and RNA and hopefully be left with nothing but strands of ~500 base pairs long segments of DNA that were bound to p75. The trick now is to sequence the DNA that I have obtained. To do this, I need a primers which are small stretches of DNA of a known sequence. These primers will bind to my DNA and enable me to enhance and then sequence the DNA. The problem is that I don't know the sequence to begin with so how can I design primers? I will do that by adding a stretch of DNA to each 500 base pair piece of DNA I isolated. Once I have done that, I can enhance each strand of DNA by PCR and then have the amplified DNA sequenced. The results will hopefully show a number of genes that were bound to the p75ICD in Vivo.

I began the first set of experiments on Monday and am having problems getting enough DNA to move on to the immunoprecipitation step. I'll keep you updated.

Cell Culture

I have a love-hate relationship with cell culture. I love it because it is easy and you can get a lot of data from one cell culture experiment if you plan correctly. I hate it because it reminds me of my time working on a dairy farm when I was in college. You can never take time off when you have cells growing in the incubator. They require constant attention in order for them to grow properly. One of the unique things about cultured cells is that most of them will stop growing once they are in contact with other cells. The ones that aren't contact inhibited are cancer cells. Anyway, I am going to be doing a FACS (fluorescence activated cell sorting) experiment using a cell line called HEK 293. It is a kidney cell line that grows well and is generally easy to care for. Unfortunately, when I went to prepare my experiment today, every well of my 6-well plate was contaminated with mold. It was fine yesterday. The only thing I did was to take out one plate of the three to look at the cells under the microscope to make sure they were growing. They were and were doing so nicely. They were about 60-70% confluent which would mean that they would be ready for viral infection today or tomorrow. Now they are all in the biohazard trash.

I am not sure what the problem is, however, I am leaning toward that 6-well plates rather than the growth media or any of the things I am doing. My reasoning is this: the cells were growing fine with no contamination issues in other growth vessels and the media shows no signs of being contaminated. I generally make enough media for the week and store it in the incubator so that any contamination will be obvious as bacteria, yeast, and mold will grow like mad in the media. Most people make up their culture media in large batches and store it in the fridge, warming it up before each use. That makes it impossible to tell if there are any contamination issues in the media (the most common source) until you have already put the media on the cells. Another reason is that I had this same problem the last time I used this batch of 6 well plates but decided that the source of contamination was elsewhere.

I am going to start another batch of 293 cells growing today and when they get ready to split, I will put half of them in a 6 well plate that I have UV irradiated (killing anything with DNA) and the other half in a non-irradiated plate. Let's hope this little experiment solves my problem. I can't keep wasting time and resources with something as silly as contaminated 293 cells.

Cochlear Implants

I love research. I love being at the bench teasing out intricate cellular interactions. Unfortunately, I spend so much time staring at the trees that I don't get to enjoy the forest very often. One of the projects of which I am a small part is an attempt to increase the efficency of cochlear implants by providing a better interface between the spiral ganglion neurons and the implant. We are experimenting with various methacrylate polymers that we are able to pattern via UV polymerization in the hopes of being able to better direct axon growth toward a target. We have had some initial success in Vitro.

This link is a quick glimpse of the forest and what we hope to improve upon.

http://www.wimp.com/hearingimpaired/

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.