Jonathan Scoville and Dr. Gregory F. Burton, Chemistry and Biochemistry

The purpose of this project was to determine the reason why the transcription factor NF-κB is not activated when immune complexes (especially those containing HIV) bind to its Fcγ-Receptor, but is activated in FDC’s when the same immune complex (IC) binds to its Fcγ-Receptor. We believed that certain molecules in B-cells bind to Fcγ-Receptor that are not present in the FDC to block activation of transcription. Our purpose was to successfully isolate these molecules in B cells, and then use them to perform the same function in CD 4 bearing T cells, HIV’s main target during infection. Our goal was to locate and isolate these molecules and identify them. If the activation of NF-κB can be inhibited by these molecules we can use them to inhibit transcription of HIV in infected individuals, decreasing the spread of the disease.

The goals of this project were to first, determine the DNA sequence of the Fcγ-Receptor on B cells and then determine the Fcγ-Receptor on FDCs so to compare them for differences. Specifically if the FDC sequence coded for an immuno-tyrosine activation motif or ITAM as it was known that the B cell sequence coded for an immuno-tyrosine inactivation motif or ITIM. In order to accomplish this goal we had to develop primers that would allow us to use polymerase chain reaction or PCR to copy and magnify the DNA sequence that codes for the Fcγ-Receptor, and then once we magnified it use sequencing techniques to sequence the DNA segment. Once we had the sequenced DNA we could determine any major variations between the FDC and B cell.

I was able to successfully generate primers for the FDC and B cell DNA sequence that code for the Fcγ-Receptor. I was unable to find any literature that had specific primers for the Fcγ-Receptor IIB which was the specific isotype of the receptor we were studying. I used primerBLAST a program offered by the NCBI website to successfully design and test primers that fulfilled our intentions. We needed to be able to isolate and magnify the receptor and most importantly the cytoplasmic domain of the receptor as that is the part that would contain the function of activating or inactivating NF-κB.

The hardest part of the project was producing the cDNA that we needed in order to test our primers and from which we ultimately amplifed our desired sequence. The first step in the process reverse transcription is isolating the desired cell. With B cells we were able to do this fairly simply and quickly. We would digest tonsils and use a Magnetic Activated Cell Sorter or MACS to do this, but FDCs were much more complicated to work with as we needed to use a Fluorescence Activated Cell Sorter or FACS which are much more complicated and delicate machines and because of the delicacy of the FDCs a more refined approach was required. I spent many hours learning how to isolate these cells. The next step in the process was learning how to isolate the RNA from the previously sorted cells. RNA is very prone to degradation and must be handled with the utmost care. This was the step that I had the most difficulty with. Learning how to successfully isolate RNA took a long time. I finally was able to overcome this hurdle by persistence, and faithfully applying correct laboratory techniques. I learned a lot from my mentor and other students who aided me in overcoming this set back.

There is still much work to be done on this project. We still need to successfully sequence and compare these two sequences. There is also the work of immuno-precipitating the Fcγ-Receptor. A process which allows us to isolate all of the molecules associated with the receptor in its progression of activating of deactivating NF-κB. I expect that this project should be completed by April 2011. I am working alongside another student from Dr. Burton’s lab and we are committed to seeing this project through and completeing it by the end of the semester. We are confident that we will have the tools to allow us to be successful in determining the role to the Fcγ-Receptor in the activation of NF-κB in FDCs.

I have learned so much from this opportunity and I am so grateful for the ORCA grant that allowed me to experience meaningful research in a positive environment. I am sure that this experience will help me as I go forward to seek a career in the field of Basic Science Research. Thank you.