INSERM UMR-S 872, Centre de Recherches des Cordeliers, Paris, France.
PLoS One. 2012;7(2):e30210. doi: 10.1371/journal.pone.0030210. Epub 2012 Feb 17.
Conditional gene deletion in specific cell populations has helped the understanding of pancreas development. Using this approach, we have shown that deleting the glucocorticoid receptor (GR) gene in pancreatic precursor cells leads to a doubled beta-cell mass. Here, we provide genetic tools that permit a temporally and spatially controlled expression of target genes in pancreatic cells using the Tetracycline inducible system. To efficiently target the Tetracycline transactivator (tTA) in specific cell populations, we generated Bacterial Artificial Chromosomes (BAC) transgenic mice expressing the improved Tetracycline transactivator (itTA) either in pancreatic progenitor cells expressing the transcription factor Pdx1 (BAC-Pdx1-itTA), or in beta cells expressing the insulin1 gene (BAC-Ins1-itTA). In the two transgenic models, itTA-mediated activation of reporter genes was efficient and subject to regulation by Doxycycline (Dox). The analysis of a tetracycline-regulated LacZ reporter gene shows that in BAC-Pdx1-itTA mice, itTA is expressed from embryonic (E) day 11.5 in all pancreatic precursor cells. In the adult pancreas, itTA is active in mature beta, delta cells and in few acinar cells. In BAC-Ins1-itTA mice tTA is active from E13.5 and is restricted to beta cells in fetal and adult pancreas. In both lines, tTA activity was suppressed by Dox treatment and re-induced after Dox removal. Using these transgenic lines, we overexpressed the GR in selective pancreatic cell populations and found that overexpression in precursor cells altered adult beta-cell fraction but not glucose tolerance. In contrast, GR overexpression in mature beta cells did not alter beta-cell fraction but impaired glucose tolerance with insufficient insulin secretion. In conclusion, these new itTA mouse models will allow fine-tuning of gene expression to investigate gene function in pancreatic biology and help us understand how glucocorticoid signaling affects on the long-term distinct aspects of beta-cell biology.
条件性基因敲除特定细胞群有助于理解胰腺发育。我们使用这种方法表明，在胰腺前体细胞中敲除糖皮质激素受体（GR）基因可导致β细胞数量增加一倍。在这里，我们提供了遗传工具，可使用四环素诱导系统在胰腺细胞中实现靶基因的时空控制表达。为了有效地将四环素激活剂（tTA）靶向特定细胞群，我们生成了表达改良型四环素激活剂（itTA）的细菌人工染色体（BAC）转基因小鼠，该激活剂在表达转录因子 Pdx1 的胰腺祖细胞（BAC-Pdx1-itTA）或在表达胰岛素 1 基因的β细胞（BAC-Ins1-itTA）中表达。在这两种转基因模型中，itTA 介导的报告基因激活是有效的，并受到多西环素（Dox）的调节。四环素调控的 LacZ 报告基因分析表明，在 BAC-Pdx1-itTA 小鼠中，itTA 从胚胎期 11.5 天开始在所有胰腺前体细胞中表达。在成年胰腺中，itTA 在成熟的β、δ细胞和少数腺泡细胞中表达。在 BAC-Ins1-itTA 小鼠中，tTA 从 E13.5 开始表达，并在胎儿和成年胰腺中局限于β细胞。在这两种系中，tTA 活性被 Dox 处理抑制，并在 Dox 去除后重新诱导。使用这些转基因系，我们在选择性胰腺细胞群中过表达了 GR，并发现前体细胞中的过表达改变了成年β细胞分数，但不影响葡萄糖耐量。相比之下，成熟β细胞中 GR 的过表达不会改变β细胞分数，但会损害葡萄糖耐量，导致胰岛素分泌不足。总之，这些新的 itTA 小鼠模型将允许精细调节基因表达，以研究基因在胰腺生物学中的功能，并帮助我们了解糖皮质激素信号如何影响β细胞生物学的长期不同方面。
