The Effects of Non-expression of the IFI44 Gene in Lung Cells by Using CRISPR-cas9
Faculty Sponsor(s)
Jennifer Grier
Location
Jereld R. Nicholson Library: Grand Avenue
Subject Area
Biochemistry
Description
The scope of our research will focus on the process of using CRISP-cas9 to insert our target gene, IFI44, into a plasmid, followed by transfection of lung cells. CRISP-cas9 is a mechanism with the potential to make precise, targeted changes to the genome of living cells. This was initially completed by inserting a generated sgRNA into a plasmid by designing specific forward and reverse primers, and isolating only complete and functional plasmids by performing a bacterial transformation and antibacterial selection. We then isolated a single colony of interest by transferring it to its own LB/ampicillin plate, identifying our plasmid using target PCR, and amplifying our entire plasmid by liquid bacterial culture. Our sgRNA insert was then confirmed to be in our plasmid via sequencing data which allowed us to transfect our lung culture cells with our plasmid of interest. Confirmation of our amplified plasmid in lung cells was done by growth in Puromycin selected plates, as the antibiotic kills 99% of untransfected cells. The results suggest that the expression of our plasmid could lead to a mutation in the lung cells.
Recommended Citation
Smith, Jacob K. and Rodriguez, Jorge, "The Effects of Non-expression of the IFI44 Gene in Lung Cells by Using CRISPR-cas9" (2017). Linfield University Student Symposium: A Celebration of Scholarship and Creative Achievement. Event. Submission 39.
https://digitalcommons.linfield.edu/symposium/2017/all/39
The Effects of Non-expression of the IFI44 Gene in Lung Cells by Using CRISPR-cas9
Jereld R. Nicholson Library: Grand Avenue
The scope of our research will focus on the process of using CRISP-cas9 to insert our target gene, IFI44, into a plasmid, followed by transfection of lung cells. CRISP-cas9 is a mechanism with the potential to make precise, targeted changes to the genome of living cells. This was initially completed by inserting a generated sgRNA into a plasmid by designing specific forward and reverse primers, and isolating only complete and functional plasmids by performing a bacterial transformation and antibacterial selection. We then isolated a single colony of interest by transferring it to its own LB/ampicillin plate, identifying our plasmid using target PCR, and amplifying our entire plasmid by liquid bacterial culture. Our sgRNA insert was then confirmed to be in our plasmid via sequencing data which allowed us to transfect our lung culture cells with our plasmid of interest. Confirmation of our amplified plasmid in lung cells was done by growth in Puromycin selected plates, as the antibiotic kills 99% of untransfected cells. The results suggest that the expression of our plasmid could lead to a mutation in the lung cells.
