Hatzoglou M, Park E, Wynshaw-Boris A, Kaung H L, Hanson R W
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
J Biol Chem. 1988 Nov 25;263(33):17798-808.
Hepatoma cells were infected with replication-incompetent murine retroviruses containing the selectable gene for amino-3'-glycosyl phosphotransferase (neo) and/or the nonselectable gene for bovine growth hormone (bGH). Expression of these genes was controlled by the promoter regulatory region of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) from the rat, which contains hormone and tissue-specific regulatory elements. Expression of the transduced PEPCK-neo gene was stimulated by Bt2cAMP and glucocorticoids and inhibited by insulin. The amount of RNA which initiated within the retroviral 5' long terminal repeat (5' LTR) was inhibited when internal promoters were present in the retroviral vector. When no internal promoter was present, expression from the 5' LTR was higher and stimulated by glucocorticoids, due to the presence of a glucocorticoid regulatory element in the 5' LTR. Infection of cells with retroviruses altered the basal expression and hormonal regulation of the endogenous PEPCK gene, but had no effect on the expression of the tyrosine aminotransferase gene, which is regulated in a similar manner by cAMP and glucocorticoids. A segment of the PEPCK promoter acted as a hormonally regulated enhancer, bringing the SV40 early promoter under the control of Bt2cAMP. A second, nonselectable gene (PEPCK-bGH), contained in the retroviral vector together with PEPCK-neo, was expressed and regulated appropriately when introduced into hepatoma cells. The proviruses were initially integrated randomly into the host cell genome, but after prolonged selection for expression of the transduced PEPCK-neo gene, cells were selected which contain a predominant site(s) of integration. Among populations of cells, however, the predominant site(s) of proviral integration was different. The selection of cells with a specific site of integration from a population was accelerated by the presence of PEPCK promoter sequences in the provirus. Despite the need to better characterize their effects on the host cell, retroviruses appear to be versatile tools for the specific introduction of regulated genes into cells.
肝癌细胞用含有氨基-3'-糖基磷酸转移酶（neo）选择基因和/或牛生长激素（bGH）非选择基因的无复制能力的鼠逆转录病毒进行感染。这些基因的表达受大鼠磷酸烯醇式丙酮酸羧激酶（GTP）（EC 4.1.1.32）（PEPCK）胞质形式基因的启动子调控区控制，该调控区含有激素和组织特异性调控元件。转导的PEPCK-neo基因的表达受Bt2cAMP和糖皮质激素刺激，受胰岛素抑制。当逆转录病毒载体中存在内部启动子时，在逆转录病毒5'长末端重复序列（5' LTR）内起始的RNA量受到抑制。当不存在内部启动子时，由于5' LTR中存在糖皮质激素调控元件，5' LTR的表达更高且受糖皮质激素刺激。用逆转录病毒感染细胞改变了内源性PEPCK基因的基础表达和激素调控，但对酪氨酸转氨酶基因的表达没有影响，该基因受cAMP和糖皮质激素以类似方式调控。PEPCK启动子的一段序列作为激素调控的增强子，使SV40早期启动子受Bt2cAMP控制。与PEPCK-neo一起包含在逆转录病毒载体中的第二个非选择基因（PEPCK-bGH），在引入肝癌细胞时能正常表达并受到适当调控。原病毒最初随机整合到宿主细胞基因组中，但在对转导的PEPCK-neo基因的表达进行长期选择后，选择出了含有主要整合位点的细胞。然而，在细胞群体中，原病毒整合的主要位点是不同的。原病毒中PEPCK启动子序列的存在加速了从群体中选择具有特定整合位点的细胞。尽管需要更好地描述它们对宿主细胞的影响，但逆转录病毒似乎是将调控基因特异性导入细胞的通用工具。
