長度4.4kb人類CYP11A1啟動子能驅動Cre重組酶在鼠腦內表現

Abstract

類固醇荷爾蒙主要於腎上腺、性腺與胎盤產生，近年已有研究發現腦部也能自行合成神經性類固醇，影響腦內的功能。在類固醇合成過程的第一步也是速率決定的步驟，是由P450膽固醇側鏈截切酶(P450scc)所催化。雖然前人利用RT-PCR技術測得此酵素的基因CYP11A1能在腦內表現，證明腦部具有新生類固醇的能力，但CYP11A1基因在腦內的表現量卻遠遠低於在腎上腺內的表現量，這使得P450scc在腦內的分佈區域和生理作用難以被確切描述。 本實驗室利用長度為4.4kb的人類CYP11A1啟動子控制Cre重組酶，產生了SCC-Cre的基因轉殖小鼠，目的是將來對類固醇生成組織內的基因做功能性的研究。我們發現Cre重組基因除了專一性表現在腎上腺與性腺，腦組織也有很好的Cre重組酶表現。在本篇研究中，我們分析Cre重組酶在腦內的活性與表現分布，結果顯示其主要分布在間腦區域的上丘腦與丘腦前端，以及中腦背部的上丘結構與頂蓋前區；在海馬迴結構中則有較弱的表現。我們進一步觀察到Cre重組基因在胚胎發育第10天的腦部開始有表現，並一直持續至出生前，其分布位置與成鼠的腦部一致。另外我們還觀察到在胚胎鼠的嗅覺上皮組織與視網膜內層有Cre表現。這些結果皆顯示轉殖的CYP11A1啟動子能驅動Cre重組酶在鼠腦內表現，且分布位置能反映目前已知的腦內內生性Cyp11a1基因的表現。鼠腦內生性Cyp11a1因表現量太低，難以被明確且直接的偵測，因此SCC-Cre小鼠提供了一個有價值的動物模式助於我們了解Cyp11a1在腦內可能的分布情形。另外， CYP11A1基因上游的4.4kb序列也是第一次被證實具有足以調控基因在腦部表現的元素。

Steroid hormones are mainly synthesized in adrenals, gonads and placenta. Recent studies have found that steroids can be de novo synthesized in brain, termed neurosteroid, to affect brain functions. The cholesterol side-chain cleavage enzyme (P450scc), encoded by CYP11A1 gene, catalyzes the first and rate-limiting step in steroid synthetic pathway. The expression of P450scc in the CNS has been detected by RT-PCR to demonstrate the steroidogenic capacity in the brain. However, the levels of CYP11A1 expression are much lower than that in adrenal glands. It is therefore difficult to identify the exact expressional pattern and also brings challenges to elucidate the physiological function of P450scc in brain. We generated a SCC-Cre transgenic mouse, in which Cre recombinase expression is under the control of the human CYP11A1 promoter. It has been shown that Cre recombinanse was specifically expressd in the adrenals and gonads. In addition, we found that the brain tissues also express Cre protein in the SCC-Cre mice. By RT-PCR analysis, the expression of Cre was identified in all regions of brain except cerebellum. High levels of Cre expression was detected in the epithalamus and anterior thalamus of the diencephalons, and superior colliculus and pretectum of the midbrain. Weak expression was also found in the hippocampal formation. The Cre recombinase activity was first present in the embryonic brain at 10.5 days postcoitum in a pattern similar to that of adult brain. The Cre expression was further observed in the olfactory epithelium and the inner layer of retina. In conclusion, the 4.4 kb of 5’flanking region of the human CYP11A1 genes contains efficient regulatory elements to drive transgene expression in the mouse brain. Our SCC-Cre transgenic mice provide a valuable animal model to understand the possible expressional pattern of CYP11A1 in the brain.