| The overall objective of this dissertation is to study the in vivo function of microsomal cytochrome P450 monooxygenases (P450s), which metabolize numerous drugs, chemical carcinogens, environmental pollutants, as well as endogenous signaling molecules such as steroid hormones and eicosanoids. The major research tool of this study involves the development of transgenic and knockout mouse models. The specific aims are (1) to study the in vivo function of NADPH-cytochrome P450 reductase (CPR) and CPR-dependent enzymes using a mouse model with a reversible hypomorphic Cpr gene; (2) to study the in vivo function of CYP2A13 with a CYP2A13- transgenic model; and (3) to generate a novel Cyp2a-2b-2f-2g-2s -null mouse model as the background strain for CYP2A13-humanized model.;In the first aim (Chapter 2), we generated a reversible Cpr-low (r-CL) mouse model, in which the globally reduced CPR expression could be reversed in an organ-specific fashion. As the first application, a new mouse model (named extrahepatic Cpr-low or "xh-CL"), which had normal CPR expression in hepatocytes, but down-regulated CPR expression in all extrahepatic tissues, was generated by crossing the r-CL model to an Alb-Cre transgenic line. By comparing the phenotypes of wild-type, r-CL and xh-CL models, we demonstrated that hepatic CPR and CPR-dependent enzymes played significant roles in maintaining normal body/major organ weights, normal blood total cholesterol levels and embryonic homeostasis. We also confirmed that restoring the hepatic function of CPR in xh-CL mice could not reverse those phenotypes such as elevated female serum testosterone/progesterone levels or female infertility;In the second aim (Chapter 3), we prepared a CYP2A13-transgenic strain with a human genomic fragment containing the full-length CYP2A13 gene, as well as CYP2B6 and CYP2F1 genes. The tissue distribution of transgenic CYP2A13 agreed well with the respiratory tract-selective expression of CYP2A13 in humans. CYP2A13 was also confirmed to be functional in transgenic mice with both 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) activity assay and genomic O6-methyl guanine analysis. However, CYP2A13-transgenic model showed significant background activities on NNK, which were presumably caused by mouse endogenous CYP2 enzymes.;In the third aim (Chapter 5), the aim was to remove a 1.4-Mb Cyp2a-2b-2f-2g-2s-gene cluster (from Cyp2f2 to Cyp2s1) on mouse Chr. 7. We succeed in inserting two loxP sites into Cyp2s1 and Cyp2f2 genes in embryonic stem (ES) cells, respectively; but failed to delete the whole cluster with in vitro electroporation of Cre-expressing plasmids. Then we tried an alternative in vivo approach based on intensive crossing with Cyp2a5 -null, Cyp2s1-null and CMVcre-transgenic mice. A 1.2-Mb Cyp2a5-Cyp2s1-cluster null mouse model was successfully generated. To our knowledge, this was the first study in which a Cre/loxP-mediated, million base-pair scale genomic deletion was obtained with an in vivo approach. |
