| Nuclear receptors are a superfamily of ligand-activated transcription factors that are homologue to steroid hormone receptors. Many researches had indicated that the coordinated expression of gene networks in developmental, cell differential, physiological, and metabolic processes can be ascribed in considerably degree to the regulation by nuclear receptor and their interaction with ligands and coregulators. Human nuclear receptor human B1 -binding factor(hB1F) was cloned and identified as a liver-enrich trasncription factor by Li when investigating the transcription regulation of hepatitis B virus(HBV) genes. It can bind specifically to the Bl region of HBV enhancer II and activate its function, thus regulating the expression of HBV gene. The amino sequence analysis indicates that hB1F is a new member of fushi tarazu factor I (Ftz-F1) subfamily, which belongs to orphan nuclear receptor. It has been named as NR5A2 according to a nomenclature system proposed in 1999.The functional investigation of hb1f and its homologue genes demonstrated that they play important roles in regulation of many liver-specific expression genes such as alpha-fetoprotein(AFP) and alpha 1-antitrypsin, as well as liver-enrich transcription factor gene hnf1 α , hnf1 β and hnf4 α .Recent studies indicated that NR5A2 can activate the expression of cholesterol 7- α hydroxylase(CYP7A1) and sterol 12- α hydroxylase(CYP8Bl),two key enzymes in bile acid biosynthesis, suggesting that NR5A2 is critical transcription factor in regulation cholesterol metabolism homeostasis. As the targeted disruption of nr5α2 led to a embryonic lethality in mouse, suggested that NR5A2 may also be important in early development. To facilitate the further functional studies of NR5A2, we plan to establish a nr5α2 transgenic mouse by using pronucleus microinjection.In the first part of this dissertation,linearized plasmid pcDNA3-hb1f which contain full length of hblf cDNA were microinjected into male pronucleus of 653 fertilized oocytes of mice. The injected eggs were implanted into the oviducts of 24 pseudo-pregnant foster mothers, of which 13 mice became pregnant and gave birth to 97 offsprings(45 , 52 ). Eleven offspring mice were identified to carry the hBlF cDNA as demonstrated by PCR analysis, out of which 7 mice were further confirmed by Southern blotting analysis. The ratio of transgene integration were 11.3% and 7.2% by PCR and Southern blotting analysis, respectively. The seven double positive micewere served as transgenic founders by mating to C57 mice to establish hb1f transgenic lineages(nominated as TGM-1, TGM-2, TGM-3, TGM-4, TGM-5, TGM-6 and TGM-7). The PCR identification results of Fl and F2 had showed that the transgenes in all 7 lineages can be transmitted stably.Four F2 of TGM-1 (2 2 ) were transgenic homozygotes verified by cross back to C57, which suggested that the lineage of nr5a2 transgenic mouse was established successfully. The RT-PCR and Western blotting analysis indicated mat among seven transgenic mouse lineages that examed, all but one lineage, TGM-2, were detected to have the expression of the transgene in liver. As drived by CMV promoter, the transgenes were also found expressing in heart, liver, lung, stomach and kidney, while not in intestine and encephalon of TGM-1In the second part of this dissertation,to learn if the expression of exogenous transgene will cause any morphologic change in transgenic mice organs,all major tissues and organs of TGM-1 were fixed with 10% formalin for pathologic analysis. No obvious pathologic changes had been seen in all examed tissues. It might be possible that the overexpression of exogenous hB1F had triggered a negative feedback response on the expression of the endogenous homologue mLRH-1, and resulted in a protect effect. In order to know which gene could be the target of this transcription factor, a comprehensive comparing study between the gene expression profiles in liver of TGM-1 and C57 was done by the genechip-based analysis technique. Totally, the expressions of 28...
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