title: Lentiviral-mediated RNAi knockdown yields a novel mouse model for studying Cyp2b function.
abstract: There are few in vivo knockout models available to study the function of Cyp2 members involved in the metabolism of endogenous and exogenous chemicals. These models may help provide insight into the cytochrome P450s (CYPs) responsible for the detoxification and activation of drugs, environmental toxicants, and endobiotics. The aim of this work is to produce a potent Cyp2b-knockdown (KD) mouse for subsequent study of Cyp2b function. We made a quintuple Cyp2b-KD mouse using lentiviral-promoted short hairpin RNA (shRNA) homologous to all five murine Cyp2b subfamily members (Cyp2b9, 2b10, 2b13, 2b19, and 2b23). The Cyp2b-KD mice are viable, fertile, and without obvious gross abnormalities except for an increase in liver weight. Expression of the three hepatic Cyp2b members, 2b9, 2b10, and 2b13, is significantly repressed as demonstrated by quantitative real-time PCR and Western blotting. The constitutive androstane receptor activator, 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), was used to determine if shRNA-mediated Cyp2b10 repression could be outcompeted by Cyp2b10 induction. TCPOBOP-treated Cyp2b-KD mice show 80-90% less Cyp2b protein expression than TCPOBOP-treated wild-type (WT) mice, demonstrating that Cyp induction does not outcompete the repressive function of the shRNA. Untreated and TCPOBOP-treated Cyp2b-KD mice are poor metabolizers of parathion compared with WT mice. Furthermore, Cyp2b-KD mice are sensitive to parathion, an organophosphate insecticide primarily metabolized by Cyp2b enzymes, when compared with WT mice. In summary, we designed an shRNA construct that repressed the expression and activity of multiple Cyp2b enzymes. We foresee that this novel Cyp2b-KD mouse model will significantly improve our understanding of the role of Cyp2b enzymes in chemical sensitivity and drug metabolism.
authors.affiliation: Environmental Toxicology Program, Clemson University, Clemson, South Carolina 29634, USA.
authors.forename: Basma
authors.initials: B
authors.lastname: Damiri
authors.forename: Eric
authors.initials: E
authors.lastname: Holle
authors.forename: Xianzhong
authors.initials: X
authors.lastname: Yu
authors.forename: William S
authors.initials: WS
authors.lastname: Baldwin
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000172:deficiency* / Q000235:genetics; D015153:Blotting, Western; D000090702:Constitutive Androstane Receptor; D000072467:Cytochrome P450 Family 2; D015536:Down-Regulation; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D007306:Insecticides / Q000378:metabolism / Q000633:toxicity; D016086:Lentivirus / Q000235:genetics*; D008099:Liver / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D010278:Parathion / Q000378:metabolism / Q000633:toxicity; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000172:deficiency* / Q000235:genetics
chemical_list: D000090702:Constitutive Androstane Receptor; D007306:Insecticides; D011725:Pyridines; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D010278:Parathion; C028474:1,4-bis(2-(3,5-dichloropyridyloxy))benzene; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C101679:Cyp2b10 protein, mouse; C033914:Cyp2b19 protein, mouse; C121503:Cyp2b9 protein, mouse; D000072467:Cytochrome P450 Family 2
references.citation: Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8033-7
references.pmid: 8396259
references.citation: Methods. 2004 Jun;33(2):164-72
references.pmid: 15121171
references.citation: J Biol Chem. 1992 Sep 25;267(27):19051-3
references.pmid: 1326542
references.citation: Toxicology. 2006 Jul 5;224(1-2):22-32
references.pmid: 16757081
references.citation: Biotechniques. 2002 Jun;32(6):1372-4, 1376, 1378-9
references.pmid: 12074169
references.citation: J Virol. 2008 Jul;82(14):7111-9
references.pmid: 18463153
references.citation: J Pharmacol Exp Ther. 2003 Dec;307(3):906-22
references.pmid: 14551287
references.citation: Genes Dev. 2001 Jan 15;15(2):188-200
references.pmid: 11157775
references.citation: Nature. 2000 Oct 19;407(6806):920-3
references.pmid: 11057673
references.citation: PLoS One. 2009 Aug 18;4(8):e6679
references.pmid: 19688097
references.citation: Proteomics. 2010 Jan;10(2):327-31
references.pmid: 19902426
references.citation: Pharmacogenetics. 2004 Jan;14(1):1-18
references.pmid: 15128046
references.citation: Toxicology. 2011 Jul 11;285(1-2):57-66
references.pmid: 21514354
references.citation: Toxicol Lett. 1999 Jun 30;107(1-3):177-87
references.pmid: 10414794
references.citation: Chem Biol Interact. 2005 Aug 15;155(3):111-28
references.pmid: 16054614
references.citation: Toxicol Appl Pharmacol. 2006 Oct 15;216(2):186-96
references.pmid: 16828826
references.citation: Mol Cell Biol. 1998 Oct;18(10):5652-8
references.pmid: 9742082
references.citation: Toxicol Appl Pharmacol. 1984 Mar 30;73(1):60-8
references.pmid: 6200955
references.citation: Biochem Pharmacol. 1988 Jan 1;37(1):71-84
references.pmid: 3276320
references.citation: Food Chem Toxicol. 2001 Aug;39(8):817-25
references.pmid: 11434989
references.citation: Toxicology. 2009 Feb 4;256(1-2):53-64
references.pmid: 19041682
references.citation: Mol Cell Biol. 2000 May;20(9):2951-8
references.pmid: 10757780
references.citation: J Biol Chem. 1996 May 17;271(20):12063-7
references.pmid: 8662637
references.citation: Drug Metab Dispos. 2009 May;37(5):1035-45
references.pmid: 19196849
references.citation: Nucleic Acids Res. 2007;35(8):e63
references.pmid: 17426130
references.citation: Science. 2002 Feb 1;295(5556):868-72
references.pmid: 11786607
references.citation: J Biol Chem. 1974 Oct 10;249(19):6302-10
references.pmid: 4153601
references.citation: Science. 2002 Oct 11;298(5592):422-4
references.pmid: 12376703
references.citation: Mech Dev. 2004 Oct;121(10):1289-97
references.pmid: 15327788
references.citation: Mol Endocrinol. 2004 Aug;18(8):1975-87
references.pmid: 15155787
references.citation: Curr Drug Metab. 2006 May;7(4):349-65
references.pmid: 16724925
references.citation: Toxicol Appl Pharmacol. 2011 May 1;252(3):259-67
references.pmid: 21376070
references.citation: Toxicology. 2006 Feb 15;219(1-3):97-105
references.pmid: 16377053
references.citation: Hepatology. 2008 Oct;48(4):1302-11
references.pmid: 18798339
references.citation: Drug Metab Dispos. 2001 Sep;29(9):1201-4
references.pmid: 11502728
references.citation: BMC Genomics. 2009 Apr 21;10:169
references.pmid: 19383150
references.citation: Am J Pathol. 1994 Aug;145(2):398-408
references.pmid: 8053497
references.citation: Gene Ther. 2002 Sep;9(17):1155-62
references.pmid: 12170379
references.citation: J Biol Chem. 2003 Apr 18;278(16):14146-52
references.pmid: 12571232
references.citation: Curr Pharmacogenomics Person Med. 2009 Jun 1;7(2):81-105
references.pmid: 20871735
references.citation: Toxicol Sci. 2007 Aug;98(2):416-26
references.pmid: 17483497
references.citation: Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6868-72
references.pmid: 1862110
references.citation: J Biol Chem. 2003 Apr 11;278(15):13480-6
references.pmid: 12566435
references.citation: Drug Metab Dispos. 2010 Sep;38(9):1582-8
references.pmid: 20573718
references.citation: J Pharmacol Exp Ther. 2004 Oct;311(1):34-43
references.pmid: 15190123
references.citation: PLoS One. 2011;6(9):e24381
references.pmid: 21931700
references.citation: Drug Metab Dispos. 2008 Jun;36(6):1080-7
references.pmid: 18339816
references.citation: Mol Cell Biol. 2001 Feb;21(4):1393-403
references.pmid: 11158324
references.citation: Pharmacol Ther. 2007 Feb;113(2):420-8
references.pmid: 17157385
references.citation: Hepatology. 2005 Jan;41(1):168-76
references.pmid: 15619241
references.citation: Curr Drug Metab. 2008 Sep;9(7):598-610
references.pmid: 18781911
references.citation: FASEB J. 2008 Dec;22(12):4096-108
references.pmid: 18716027
references.citation: J Pharmacol Exp Ther. 1985 Mar;232(3):624-8
references.pmid: 2579231
references.citation: BMC Res Notes. 2010 Mar 03;3:53
references.pmid: 20199660
references.citation: Arch Biochem Biophys. 1999 Sep 1;369(1):89-99
references.pmid: 10462443
references.citation: Curr Protoc Mol Biol. 2002 Nov;Chapter 16:Unit 16.22
references.pmid: 18265303
references.citation: Physiol Genomics. 2007 Oct 22;31(2):159-73
references.pmid: 17684037
references.citation: J Pharmacol Exp Ther. 1998 Sep;286(3):1253-9
references.pmid: 9732386
references.citation: J Clin Invest. 2007 Nov;117(11):3583-92
references.pmid: 17975676
references.citation: Nucleic Acids Res. 2002 Apr 15;30(8):1757-66
references.pmid: 11937629
references.citation: Drug Metab Dispos. 2007 Feb;35(2):189-93
references.pmid: 17079358
references.citation: Nucleic Acids Res. 2011 Jan;39(Database issue):D835-41
references.pmid: 21062809
references.citation: J Toxicol Environ Health A. 2005 Dec 10;68(23-24):2187-205
references.pmid: 16326433
references.citation: Curr Drug Metab. 2006 May;7(4):397-409
references.pmid: 16724929