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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Conditional transformation of a pancreatic β-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene
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Conditional transformation of a pancreatic β-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene

机译：来自表达四环素调节致癌基因的转基因小鼠的胰腺β细胞系的条件转化

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摘要

Conditional oncogene expression in transgenic mice is of interest for studying the oncoprotein requirements during tumorigenesis and for deriving cell lines that can be induced to undergo growth arrest and enhance their differentiated functions. We utilized the bacterial tetracycline (Tet)-resistance operon regulatory system (tet) from Tn10 of Escherichia coli to control simian virus 40 (SV40) large tumor (T) antigen (TAg) gene expression and to generate conditionally transformed pancreatic β cells in transgenic mice. A fusion protein containing the tet represser (tetR) and the activating domain of the herpes simplex virus protein VP16, which converts the represser into a transcription activator, was produced in β cells of transgenic mice under control of the insulin promoter. In a separate lineage of transgenic mice, the TAg gene was introduced under control of a tandem array of tet operator sequences and a minimal promoter, which by itself is not sufficient for gene expression. Mice from the two lineages were then crossed to generate double-transgenic mice. Expression of the tetR fusion protein in β cells activated TAg transcription, resulting in the development of β-cell tumors. Tumors arising in the absence of Tet were cultured to derive a stable β-cell line. Cell incubation in the presence of Tet led to inhibition of proliferation, as shown by decreased BrdUrd and [~3H]thymidine incorporation. The Tet derivative anhydrotetracycline showed a 100-fold stronger inhibition compared with Tet. When administered in vivo, Tet efficiently inhibited β-cell proliferation. These findings indicate that transformed β cells selected for growth during a tumorigenesis process in vivo maintain a dependence on the continuous presence of the TAg oncoprotein for their proliferation. This system provides an approach for generation of β-cell lines for cell therapy of diabetes as well as conditionally transformed cell lines from other cell types of interest.
机译：转基因小鼠中有条件的癌基因表达对于研究肿瘤发生过程中对癌蛋白的需求以及衍生出可诱导其经历生长停滞并增强其分化功能的细胞系具有重要意义。我们利用大肠杆菌Tn10的细菌四环素（Tet）抗性操纵子调控系统（tet）控制猿猴病毒40（SV40）大肿瘤（T）抗原（TAg）基因表达，并在转基因中产生条件转化的胰腺β细胞老鼠。在胰岛素启动子的控制下，在转基因小鼠的β细胞中产生了包含tet阻遏物（tetR）和单纯疱疹病毒蛋白VP16的激活域的融合蛋白，该融合蛋白将阻遏物转化为转录激活因子。在一个单独的转基因小鼠谱系中，TAt基因在tet操纵基因序列和最小启动子的串联阵列的控制下被引入，这本身不足以表达基因。然后将来自两个谱系的小鼠杂交以产生双转基因小鼠。 tetR融合蛋白在β细胞中的表达激活了TAg转录，从而导致β细胞肿瘤的发展。培养不存在Tet的肿瘤，以培养出稳定的β细胞系。在Tet存在下进行细胞孵育可抑制增殖，如BrdUrd和[〜3H]胸腺嘧啶核苷掺入减少所示。与Tet相比，Tet衍生物脱水四环素的抑制作用强100倍。当在体内给药时，Tet有效抑制β细胞增殖。这些发现表明，在体内肿瘤发生过程中被选择用于生长的转化的β细胞维持对TAg癌蛋白持续存在的依赖以使其增殖。该系统提供了一种用于产生用于糖尿病的细胞疗法的β细胞系以及来自其他目的细胞类型的有条件转化的细胞系的方法。

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