Presented at the Annual Biomedical Research Conference for Minority Students (ABRCMS)
Dr. Estefany Rios Guzman; Northwestern University, Feinberg School of Medicine
Margarita Rzhetskaya; Northwestern University, Feinberg School of Medicine
Dr. Judd F. Hultquist; Northwestern University, Feinberg School of Medicine
With no current vaccine for Human Immunodeficiency Virus (HIV), it is crucial to study what factors influence how HIV infects host cells to develop new strategies and therapies to combat this pandemic. Cleavage and polyadenylation specificity factor 6 (CPSF6) is an HIV host factor known to influence HIV infectivity and trafficking early in the infection. Two isoforms of CPSF6 exist, but the true difference between the two are currently unknown. This investigation strives to discover the difference between the two isoforms in regard to the innate immune response. Initially, we require a way to overexpress CPSF6 in mammalian cells to observe the antiviral response in HIV infection. To make a CPSF6-containing vector, we prepared a pLVX-TetOne-Puro-Cter3xFlag empty vector through E. coli transformation and plasmid purification, restriction enzyme digest, and then gel electrophoresis to confirm successful plasmid linearization. Post-purification, two inserts of the CPSF6 gene were then assembled into the empty vector using Gibson Assembly reactions. With this assembled construct, repetition of all of the aforementioned steps were performed to confirm the successful creation of isoform 2 of CPSF6. To create isoform 1 of CPSF6, polymerase chain reaction (PCR) was used to isolate and later delete a specific exon in CPSF6 from isoform 2. Once successful creation of isoform 1 and 2 were confirmed, isoform 2 was expressed in mammalian cells and were tested for their antiviral gene expression by Western blot. Future research will include expressing isoform 1 in mammalian cells and testing their antiviral gene expression by Western blot. I also hope to test both plasmid's infectivity for HIV by flow cytometry. Ultimately, this experiment will lead to a greater understanding of CPSF6’s role in the innate immune response in HIV infection.
The Impact of CPSF6 on the Innate Immune Response to HIV Infection.
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