The ELISA has been coming out pretty well for the last two weeks. Here's a bit of background information about the uses of this assay in this particular experiment.
As I mentioned before, alpha-Gal (abbrev. Gal) is a sugar found on natural antibody producing B cells, binding onto the BSA molecule. Gal is found in mice and pigs, but not in humans, which makes the field of xenotransplantion (transplanting organs between different species) very difficult, as the presence of Gal on a transplanted organ can trigger an immune response because humans have anti-Gal antibodies. This is one of the many barriers to xenotransplantation. However, if the barriers can be overcome, xenotransplantation could open up possibilities of almost endless organ transplant options, especially from pigs to humans. This new source of organs could curb the waiting list for tranplants dramatically, and save millions of lives of people who are dying just waiting for an transplant option to open. To study this, we use Gal KO mice (that are abnormal in that they don't have any Gal) and then give them a bone marrow transplant from a normal Gal positive mouse. The ELISAs that I have been doing have been testing for anti-Gal antibodies - that is, an immune response to Gal. If the mice are tolerant, there is no immune response - therefore, there are no anti-Gal antibodies seen in the assay. These tolerant mice are chimeras.
Thursday, July 24, 2008
Wednesday, July 16, 2008
Week Five
This week has been full of collecting data through the experiment known as the ELISA. ELISA stands for Enzyme-Linked ImmunoSorbent Assay. It's used for looking for antibodies. Here, the ELISA data can be used along with the Elispot data and the FACS data to prove that there is indeed an immune response taking place where they are supposed to.
On Monday, I did an ELISA looking at the immune responses with the sera of a few different mice in different dilutions, so as to determine which of these seras and at which particular dilution is good to serve as a control for the next few ELISAs.
On Tuesday, the ELISA utilized the sera of mice that were part of the same takedown that yielded the Elispot and FACS data on the spleen and peritoneal cavity over the last two weeks. Wednesday was a repeat of the same ELISA to check that the results are correct. Thursday, I did an ELISA using the sera from the other 3 mice part of the same takedown. With a combination of all these results, we're going to lab meeting to present the data and look for ideas and input from the other lab fellows on what to do next.
On Monday, I did an ELISA looking at the immune responses with the sera of a few different mice in different dilutions, so as to determine which of these seras and at which particular dilution is good to serve as a control for the next few ELISAs.
On Tuesday, the ELISA utilized the sera of mice that were part of the same takedown that yielded the Elispot and FACS data on the spleen and peritoneal cavity over the last two weeks. Wednesday was a repeat of the same ELISA to check that the results are correct. Thursday, I did an ELISA using the sera from the other 3 mice part of the same takedown. With a combination of all these results, we're going to lab meeting to present the data and look for ideas and input from the other lab fellows on what to do next.
Tuesday, July 15, 2008
Week Four
As the weeks go by, much more of the analysis that comes with the experimentation that is taking place here at the lab is making sense to me. Any time that a summer student has a question, especially if it is asked for clarity on a particular subject of the experiment being done, the fellows here are more than glad to sit down for a few minutes and review many more concepts of immunology and their associations and implications in this field of study. For example, Carrie has a new student this week, and I've been able to listen to a few of her explanations, from the basics of the immune system to the different methods to how exactly a gene is "knocked out". I've gradually been absorbing more and more information, and it definitely gets easier!
An example of a concept that I've begun to grasp - A knocked out gene means that although it is still present, it is no longer expressed. One way to knockout a gene is to use a method of homologous recombination, by basically providing another pathway (a vector) for the mouse genome to follow for a specific gene, which essentially makes the gene usually in this replaced spot no longer expressed. To target and quantify those genes that follow this process of homologous recombination - that is, the genes that forego their original path and successfully take up the vector - a neomycin resistance gene can be used in the vector. This way, you ensure which mouse genomes have taken up the vector, and use only these mice for breeding chimeras. Another method is transgenic mice, which is a less specifically targeted approach to this.
Why are gene knockouts important? By knocking out specific genes, a researcher can enlarge the possibilities of research on various topics almost endlessly.
This week for experiments, I've seen another Elispot done on the peritoneal cavity cells of the mice taken down last week. Tuesday, we went out to dinner in Charlestown with Mr. Palm, which was a lot of fun! I've also been helping map and catalog more of the in vivo imaging data which Ellen and Tosh are working on. And on Friday, I got to do my first assay by myself! It's called an Elisa, and I'll be doing many more of these for the next few weeks.
An example of a concept that I've begun to grasp - A knocked out gene means that although it is still present, it is no longer expressed. One way to knockout a gene is to use a method of homologous recombination, by basically providing another pathway (a vector) for the mouse genome to follow for a specific gene, which essentially makes the gene usually in this replaced spot no longer expressed. To target and quantify those genes that follow this process of homologous recombination - that is, the genes that forego their original path and successfully take up the vector - a neomycin resistance gene can be used in the vector. This way, you ensure which mouse genomes have taken up the vector, and use only these mice for breeding chimeras. Another method is transgenic mice, which is a less specifically targeted approach to this.
Why are gene knockouts important? By knocking out specific genes, a researcher can enlarge the possibilities of research on various topics almost endlessly.
This week for experiments, I've seen another Elispot done on the peritoneal cavity cells of the mice taken down last week. Tuesday, we went out to dinner in Charlestown with Mr. Palm, which was a lot of fun! I've also been helping map and catalog more of the in vivo imaging data which Ellen and Tosh are working on. And on Friday, I got to do my first assay by myself! It's called an Elisa, and I'll be doing many more of these for the next few weeks.
Sunday, July 6, 2008
Week Three
This week has been great for lab results. Earlier in the week, Josh enlisted the help of Meredith to perform a takedown of ten mice, testing for differences between the results of chimeras, normal B6 mice, and a naive Gal KO mouse (in which the Gal antibodies commonly present in mice and pigs are knocked out). Tests were done using the spleen and the fluid from the peritoneal cavity of the mice. According to the hypotheses for this takedown, there should have been a loss of B cells that bind Gal in the FACS analysis, as well as a loss of Gal antibody secreting cells in the Elispot treatment. These were predicted in the Gal KO mouse as well as the control B6. Both the FACS and the Elispot yielded clear results. I've been helping with the centrifuging, cell counting, and FACS and flow-jo analyses on each part of the experiment this week, and I've been learning new techniques and protocols every day.
Another thing that came in this week was a newer version of the toxin product that needed to be tested before using it on mouse injections for bone marrow transplants and irradiation to induce chimerism. The product worked well, so it is safe to say that a switch can be made from the old product to the new one.
Meanwhile, Ellen and her postdoc Tosh have been continuing to conduct their research on tumor cells in mice, using the GFP (green fluorescent protein) and identifying it in the pictures taken through in vivo imaging down at a lab at the MGH main campus. Ellen and I have been helping to assemble the data maps into easily readable files on the computer.
Another thing that came in this week was a newer version of the toxin product that needed to be tested before using it on mouse injections for bone marrow transplants and irradiation to induce chimerism. The product worked well, so it is safe to say that a switch can be made from the old product to the new one.
Meanwhile, Ellen and her postdoc Tosh have been continuing to conduct their research on tumor cells in mice, using the GFP (green fluorescent protein) and identifying it in the pictures taken through in vivo imaging down at a lab at the MGH main campus. Ellen and I have been helping to assemble the data maps into easily readable files on the computer.
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