Greenberg N M, Reding T V, Duffy T, Rosen J M
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Mol Endocrinol. 1991 Oct;5(10):1504-12. doi: 10.1210/mend-5-10-1504.
Previous studies have demonstrated that the entire rat beta-casein (R beta C) gene and a -524/+490 R beta C fragment-chloramphenicol acetyltransferase (CAT) fusion gene are expressed preferentially in the mammary gland of transgenic mice in a developmentally regulated fashion. However, transgene expression was infrequent, less than 1% of that observed for the endogenous gene, and varied as much as 500-fold, presumably due to the site of chromosomal integration. To determine whether a heterologous hormone-responsive enhancer could be used to increase both the level and frequency of expression in the mammary gland, a fragment derived from the mouse mammary tumor virus long terminal repeat containing four hormone response elements (HREs) was inserted into the R beta C promoter at a site not known to contain transcriptional regulatory elements. Transgenic mice generated which carried HRE-enhanced R beta C-CAT fusion genes expressed CAT activity in the mammary glands of all founder lines examined at levels that were on average 13-fold greater than for lines generated with similar constructs not carrying HREs. In the highest expressing line, the level of HRE-enhanced transgene expression was found to be developmentally regulated, increasing 14-fold in the mammary gland from virgin to day 10 of lactation. In this line, expression was also observed in the thymus and spleen; however, the level of CAT activity was 4-fold lower than in the mammary gland and was not developmentally regulated. In adrenalectomized mice, the administration of dexamethasone stimulated CAT expression in the mammary gland but not in the thymus and spleen. These studies demonstrate that in the context of the R beta C promoter, the HRE functions in the mammary gland to increase both the frequency and level of transgene expression.
先前的研究表明，完整的大鼠β-酪蛋白（RβC）基因以及一个-524/+490 RβC片段-氯霉素乙酰转移酶（CAT）融合基因，在转基因小鼠的乳腺中以发育调控的方式优先表达。然而，转基因表达很少见，不到内源基因表达量的1%，并且变化幅度高达500倍，推测这是由于染色体整合位点的原因。为了确定一个异源激素反应增强子是否可用于提高乳腺中的表达水平和频率，将一个来自小鼠乳腺肿瘤病毒长末端重复序列、含有四个激素反应元件（HREs）的片段，插入到RβC启动子中一个未知含有转录调控元件的位点。携带HRE增强的RβC-CAT融合基因的转基因小鼠，在所有检测的奠基者品系的乳腺中都表达CAT活性，其表达水平平均比携带不含HREs的类似构建体产生的品系高13倍。在表达水平最高的品系中，发现HRE增强的转基因表达水平受发育调控，在乳腺中从处女期到泌乳第10天增加了14倍。在这个品系中，在胸腺和脾脏中也观察到了表达；然而，CAT活性水平比在乳腺中低4倍，并且不受发育调控。在肾上腺切除的小鼠中，给予地塞米松可刺激乳腺中的CAT表达，但不刺激胸腺和脾脏中的表达。这些研究表明，在RβC启动子的背景下，HRE在乳腺中发挥作用，可提高转基因表达的频率和水平。
