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.