NDLI: Repression of multiple CYP2D genes in mouse primary hepatocytes with a single siRNA construct

Content Provider	Springer Nature Link
Author	Elraghy, Omaima Baldwin, William S.
Copyright Year	2014
Abstract	The Cyp2d subfamily is the second most abun-dant subfamily of hepatic drug-metabolizing CYPs. In mice, there are nine Cyp2d members that are believed to have redundant catalytic activity. We are testing and optimizing the ability of one short interfering RNA (siRNA) construct to knockdown the expression of multiple mouse Cyp2ds in primary hepatocytes. Expression of Cyp2d10, Cyp2d11, Cyp2d22, and Cyp2d26 was observed in the primary male mouse hepatocytes. Cyp2d9, which is male-specific and growth hormone-dependent, was not expressed in male primary hepatocytes, potentially because of its dependence on pulsatile growth hormone release from the anterior pituitary. Several different siRNAs at different concentrations and with different reagents were used to knockdown Cyp2d expression. siRNA constructs designed to repress only one construct often mildly repressed several Cyp2d isoforms. A construct designed to knockdown every Cyp2d isoform provided the best results, especially when incubated with transfection reagents designed specifically for primary cell culture. Interestingly, a construct designed to knockdown all Cyp2d isoforms, except Cyp2d10, caused a 2.5× increase in Cyp2d10 expression, presumably because of a compensatory response. However, while RNA expression is repressed 24 h after siRNA treatment, associated changes in Cyp2d-mediated metabolism are tenuous. Overall, this study provides data on the expression of murine Cyp2ds in primary cell lines, valuable information on designing siRNAs for silencing multiple murine CYPs, and potential pros and cons of using siRNA as a tool for repressing Cyp2d and estimating Cyp2d’s role in murine xenobiotic metabolism.
Starting Page	9
Ending Page	14
Page Count	6
File Format	PDF
ISSN	10712690
Journal	In Vitro Cellular & Developmental Biology - Animal
Volume Number	51
Issue Number	1
e-ISSN	1543706X
Language	English
Publisher	Springer US
Publisher Date	2014-08-15
Publisher Place	Boston
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	hepatocytes In vitro RNAi P450 Cyp2d Cell Biology Developmental Biology Stem Cells Cell Culture Animal Genetics and Genomics
Content Type	Text
Resource Type	Article
Subject	Cell Biology Developmental Biology Medicine

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