Presentation Title

Cloning of Insertion Cassette for Direct Homologous Recombination in Hypoxanthine-Guanine Phosphoribosyltransferase Gene

Faculty Mentor

Dong Sung An, Olivier Pernet

Start Date

18-11-2017 10:00 AM

End Date

18-11-2017 11:00 AM

Location

BSC-Ursa Minor 57

Session

Poster 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Human Immunodeficiency Virus (HIV) targets host immune cells, thus weakening the immune system of patients. Suppression of viral load through a cocktail of drugs has allowed HIV patients to live extended lifespans. However, with the advance of endonuclease gene edition, more attention has been placed upon the possibility of altering the patient’s genes to combat HIV/AIDS. We hypothesized that Hypoxanthine-Guanine Phosphoribosyltransferase (HPRT) gene cleavage with a Cas9 endonuclease system previously developed in our lab can lead to Direct Homologous Recombination (DHR) and therefore be subverted to insert a DNA homologous template into the cleaved region. Our goal was to build a template that contained anti-HIV genes. This anti-HIV therapeutic gene cassette would therefore be flanked by DNA homologous to the HPRT Exon 3 gene and be obtained through cloning and PCR mutagenesis. We amplified the Cas9 target HPRT Exon 3 region from genomic DNA and subsequently inserted genes of interest at cleavage sites. We produced three vectors containing different genes of interest, including anti-HIV genes. Due to the homology arms, these expression cassettes would act as the DNA template for homologous recombination in the HPRT Exon 3 region after Cas9 system specific cleavage. Through DHR, therapeutic HIV genes could be incorporated into hematopoietic stem cells, which have the ability to proliferate and provide continuous immunity to HIV/AIDS, thus offering life-long remission of this disease without the need of unremitting drugs.

Summary of research results to be presented

We successfully created an insertion cassette. This cassette is designed to trigger homologous recombination in HPRT gene. With overlap PCR, we created an NheI restriction site on the FG12 HPRT Exon 3 plasmid allowing for insertion of desired genes. Genes of interest necessitated the addition of NheI at 3' ends for ligation with FG12 HPRT Exon 3 plasmids. The presence of NheI site within 1α-ZsGreen required partial digestion to isolate the desired gene.

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Nov 18th, 10:00 AM Nov 18th, 11:00 AM

Cloning of Insertion Cassette for Direct Homologous Recombination in Hypoxanthine-Guanine Phosphoribosyltransferase Gene

BSC-Ursa Minor 57

Human Immunodeficiency Virus (HIV) targets host immune cells, thus weakening the immune system of patients. Suppression of viral load through a cocktail of drugs has allowed HIV patients to live extended lifespans. However, with the advance of endonuclease gene edition, more attention has been placed upon the possibility of altering the patient’s genes to combat HIV/AIDS. We hypothesized that Hypoxanthine-Guanine Phosphoribosyltransferase (HPRT) gene cleavage with a Cas9 endonuclease system previously developed in our lab can lead to Direct Homologous Recombination (DHR) and therefore be subverted to insert a DNA homologous template into the cleaved region. Our goal was to build a template that contained anti-HIV genes. This anti-HIV therapeutic gene cassette would therefore be flanked by DNA homologous to the HPRT Exon 3 gene and be obtained through cloning and PCR mutagenesis. We amplified the Cas9 target HPRT Exon 3 region from genomic DNA and subsequently inserted genes of interest at cleavage sites. We produced three vectors containing different genes of interest, including anti-HIV genes. Due to the homology arms, these expression cassettes would act as the DNA template for homologous recombination in the HPRT Exon 3 region after Cas9 system specific cleavage. Through DHR, therapeutic HIV genes could be incorporated into hematopoietic stem cells, which have the ability to proliferate and provide continuous immunity to HIV/AIDS, thus offering life-long remission of this disease without the need of unremitting drugs.