Characterization of the DNA elements that regulate the expression of CYP11A1 promoter in mouse olfactory epithelium

• Main Authors: Kai-Hao Liang, 梁凱豪
• Other Authors: Meng-Chun Hu
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/65057000411803513176

碩士 === 國立臺灣大學 === 生理學研究所 === 101 === Abstract Steroid hormones are mainly synthesized in adrenal cortex, gonads and placenta. Recent studies indicate that they are also produced in CNS. These so-called neurosteroids do affect physiological functions including neural survival, myelination, neurogenesis, etc. CYP11A1 encodes P450scc (cytochrome P450 side chain cleavage), which catalyzes the first and the rate-limiting step during steroidogenesis that changes cholesterol into pregnenolone. Thus, CYP11A1 plays an important part in steroidogenesis. CYP11A1 can be expressed in steroidogenic tissue, but at low level in brain. This makes CYP11A1 difficult to be detected and studied in brain. We tried to investigate the regulation and distribution of CYP11A1 by transgenic mice and proved that 4.4 kb length of CYP11A1 promoter is able to promote Cre recombinase expression in brain. Furthermore, we have curtailed the range which may contain the potential DNA elements in CYP11A1 promoter. In this thesis, we spliced the potential regulatory sequence into short segments and incubated them with mouse olfactory epithelium nuclear extracts for electrophoretic mobility shift assay (EMSA). Our results indicate that the binding sequences of olfactory epithelium nuclear molecules do exist in CYP11A1 promoter and may play important roles in its neural regulation. Since using transgenic mice for promoter research costs time and money, we are looking for some other in vitro transgenic system. Therefore, we constructed the retina electroporation system. We sent CYP11A1 promoter with Cre recombinase as the reporter gene in to retina tissue dissociated from mice cub. The results showed that although the retina electroporation system is able to transfect plasmids in to retina tissue, it still cannot help the weak promoter to trigger the reporter gene expression. In summary, this system is suitable for promoter studies except CYP11A1 promoter.