Date of Award
Spring 5-18-2020
Degree Type
Thesis
Degree Name
MS Biology
Department
Biology
Advisor
Jane Ko, Ph.D.
Committee Member
Daniel Brian Nichols, Ph.D.
Committee Member
Tin-Chun Chu, Ph.D.
Keywords
RACK1, MOR, opioids, CRISPR, Gene Expression, Gene Regulation, Knockout, Cell Line, Molecular Biology
Abstract
The µ-opioid receptor (MOR) is an important regulator of nociception, bearing the largest role in analgesia of all opioid receptors. This function has made MOR an effective drug-target for the clinical treatment of pain. However, opioid drugs such as morphine and fentanyl are highly addictive, and prolonged abuse can be fatal due to respiratory depression. A key determinant in the cellular and systemic responses to opioids is MOR. At the transcriptional level, MOR is positively regulated by the transcription factors, including poly-C binding protein 1 (PCBP1). Using the two-hybrid screening, PCBP1 was found to interact with receptor for activated C kinase (RACK1). RACK1 expression was negatively correlated with MOR expression. Here, the CRISPR technique (CRISPR-Cas9n) was used to create the deletion of the RACK1gene using human NMB neuronal cells. Screening for the deletion of RACK1 in candidate clones detected only RACK1+/- cells, but no RACK1-/- cells. Preliminary data showed that partial deletion of the RACK1 gene successfully decreased RACK1 mRNA expression and increased MOR mRNA expression in RACK1+/- cells. The stability of RACK1+/- cells was further examined. Partial deletion of the RACK1 gene, as well as alterations in mRNA expression of RACK1 and MOR, were consistently detected across a period of about five months. These preliminary results show that RACK1 can negatively regulate MOR expression.
Recommended Citation
Missall, Roy, "Generation and Characterization of a RACK1 Knockout Cell Line" (2020). Seton Hall University Dissertations and Theses (ETDs). 2744.
https://scholarship.shu.edu/dissertations/2744
