Date of Award
Summer 6-2021
Degree Type
Thesis
Degree Name
MS Microbiology
Department
Biology
Advisor
Jane Ko, Ph.D.
Committee Member
Tinchun Chu, Ph.D.
Committee Member
Heping Zhou, Ph.D.
Keywords
Opioid Receptor, RACK1, PCBP1, CRISPR-Cas9n, Genomic DNA, mRNA, Protein
Abstract
The µ-opioid receptor, MOR, is the primary target for exogenous and endogenous analgesics, such as morphine or oxycodone, used to relieve pain following physical trauma or invasive procedures. When opioid analgesics are utilized extensively, patients can develop tolerance heightening the risk of respiratory depression and subsequently death. MOR gene expression is known to be regulated by several transcription factors including poly-C binding protein 1, PCBP1, which also interacts with receptor for activated C kinase, RACK1. Using overexpression and siRNA RACK1 was shown to be inversely related to MOR expression. To further investigate the relationship of RACK1 and MOR expression, RACK1 knock-outs of human NMB cells were generated using CRISPR-Cas9n. Genomic DNA screening showed the presence of RACK1 partial knock-out cells (RACK1+/-). The allelic presence of RACK1 was examined using the semi-quantitative PCR method with primers specific for RACK1 and an internal standard, which validated the presence of RACK1+/- lines verses RACK1+/+ with a 66% and 45% decrease in RACK1+/- cell lines, RACK1-A and RACK1-B correspondingly, compared to the WT as 100%. Following, mRNA expression of RACK1 and MOR showed an inverse correlation to MOR expression which were stable across a span of nearly 5 months, with an average decrease of 58% in RACK1 expression and increase of 139% in MOR expression in RACK1-B. Similar results were found for RACK1-A with decreased RACK1 mRNA expression at an average of about 59% of the WT, and elevated MOR expression of 170% of the WT. Western blot analysis revealed decreased levels of RACK1 in RACK1+/- lines compared to the WT control with average expression of 52% and 61% for RACK1-A and RACK1-B correspondingly. Additionally, there was no significant effect on growth rate in RACK1-A and RACK1-B compared to the control. This study supports the negative regulation of RACK1 on MOR gene expression through partial RACK1 knock-out cell lines generated by the CRISPR-Cas9n method.
Recommended Citation
Modi, Zil, "Investigation of RACK1 effect on MOR Expression Using CRISPR Technology" (2021). Seton Hall University Dissertations and Theses (ETDs). 2904.
https://scholarship.shu.edu/dissertations/2904
