Date of Award

8-2011

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Biological Sciences

Advisor

Baldwin, William S

Committee Member

Bain , Lisa

Committee Member

Hughes , Thomas

Committee Member

McNealy , Tamara

Abstract

The CYP2D subfamily is the second most important subfamily of hepatic drug metabolizing CYPs. In mouse, there are nine CYP2D subfamily members, while humans have only one highly polymorphic CYP2D member, CYP2D6. siRNA was used to repress the expression of multiple isoforms of mouse CYP2D in primary mouse hepatocytes with one siRNA construct. Successful knockdown of CYP function by RNAi may allow for further study of CYP2D function. Q-PCR demonstrated that male CD-1 mouse primary hepatocytes express Cyp2d10, Cyp2d11, Cyp2d22, and Cyp2d26; however the male specific and growth hormone-dependent, Cyp2d9, was not detected. Four different siRNAs were designed; Cyp2d-KD1 and Cyp2d-KD2 target multiple CYPs while Cyp2d10-Design A and Cyp2d10-Design B only target Cyp2d10. 90% transfection efficiency with minimal cellular toxicity was achieved when Mirus-TKO transfection reagent was used. qPCR demonstrated that the construct designed to knockdown every CYP2D isoform, siRNA-KD1, showed knockdown efficiency of 50-70% and these results were reproducible with all four CYP2D members tested. Interestingly, a construct designed to knockdown all CYP2D isoforms except Cyp2d10, siRNA-KD2, induced Cyp2d10 expression 2.5-fold, presumably because of a compensatory response. Testosterone hydroxylation activity, tamoxifen metabolism and CYP2D activity were tested in scrambled and Cyp2d-KD1 siRNA treated hepatocytes using TLC, HPLC and CYP2D6-Glo(tm) Screening Systems respectively. No metabolites differences were observed. Overall, this study may provide a new tool for determining CYP2D's role in xenobiotic metabolism, valuable information on how to design siRNA studies for silencing CYP subfamilies and ultimately may help in silencing mouse CYP2D's within a humanized CYP2D6 mouse model.

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