The expression and regulation of CYP11A1 gene in the brain

• Main Authors: Yen-Feng Chiang, 江衍楓
• Other Authors: Meng-Chun Hu
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/99288049058782218755

碩士 === 臺灣大學 === 生理學研究所 === 95 === Recent studies indicated that neurosteroids play an important role in the physiological function of brain. The first and rate-limiting step of the steroid synthesis pathway is catalyzed by the cytochrome P450 side chain cleavage enzyme (P450scc) which is encoded by CYP11A1 gene. It is difficult to study because of the low expression levels. The expression pattern and transcriptional regulation of the CYP11A1 gene in nervous system is largely unknown. Our previous SCC-Cre/R26R transgenic model showed that the human CYP11A1 4.4 kb promoter can drive the transgene Cre recombinase expressed in specific regions of the mice brain. In order to verify the endogenous gene expression in these brain regions, we performed immunohistochemistry to detect the presence of P450scc in mice brain. Our result showed that P450scc expression was observed in hippocampus, habenular nucleus, neuron nucleus of thalamus and hypothalamus, and cerebral neocortex. The colocalization of P450scc and NeuN demonstrated that most of P450scc exist in neuron cell in these regions. In order to clarify the transcriptional regulation of CYP11A1 gene, established the transient SCC-iCre transgenic mice model by using R26R system to study the promoter activity of human CYP11A1 gene in the brain. The 4.4 kb or 2.7 kb of 5’-flanking region of the human CYP11A1 gene was fused to the Cre recombinase and the transgenes were injected to the ROSA26 heterozygote pronuclei by microinjection. The transgenic embryos were collected at embryonic day 14.5 and assayed for the activity of Cre recombinase by whole-mount X-gal staining. The results displayed that both 4.4 kb and 2.7 kb promoter can drive the Cre expression in the embryonic brain, but the distinct difference in expression pattern between the two promoters is observed. The activity of Cre recombinase is predominantly detected in the midbrain, diencephalon and olfactory bulb in the SCC4.4-iCre transgenic brain. However, it only can be observed in the caudal part of the midbrain in the SCC2.7-iCre brains. The results suggest that CYP11A1 gene expression could be differentially regulated in different regions of the brain. Liver receptor homolog-1 (LRH-1) is one member of the nuclear receptor family. It has been show that LRH-1 is highly expressed in the granulose cells and luteal cells of the ovary, but its physiological function is not certain. In order to explore the potential role of LRH-1 in the ovary, we construct the EGFP-mLRH11~240 fusion protein which is driven by the human CYP11A1 promoter to generate the SCC-ELRHdn transgenic mice. The mLRH11~240 has the dominant-negative effect on the transactivity of mLRH-1. The transgene expression are detected by RT-PCR analysis in the transgenic mice. In addition, the potential target genes Cyp11a1 and Cyp19 are slightly down-regulated. However, the transgenic animals do not exhibit abnormalities in ovary development and fertility.