Developing tTA transgenic rats for inducible and reversible gene expression.
Developing tTA transgenic rats for inducible and reversible gene expression.
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开发TTA转基因大鼠，用于诱导和可逆基因表达。
DOI：
10.7150/ijbs.5.171
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发表时间：
2009
影响因子：
9.2
通讯作者：
Xia XG
中科院分区：
文献类型：
--
作者：
Zhou H;Huang C;Yang M;Landel CP;Xia PY;Liu YJ;Xia XG
为了开发用于在大鼠中有条件表达所需基因的转基因品系，我们产生了携带四环素控制的反式激活因子（tTA）基因的转基因大鼠的几个品系。使用一个充满活力的，无处不在的启动子来驱动tTA转基因，我们获得了广泛的tTA在各种组织中的表达。tTA的表达足以强烈激活其报告基因，但低于毒性阈值。我们研究了多西环素（Dox）调控的基因表达在转基因大鼠的动力学。在这两个传输线，tTA介导的报告基因的激活是完全受调节的Dox。Dox剂量依赖性地抑制tTA激活的基因表达。Dox效应的洗脱时间呈剂量依赖性。我们测试了一个复杂的方案的Dox管理，以确定最佳的有效性和洗脱持续时间。Dox以高剂量（500 μg/ml在饮用水中）给药两天以达到有效浓度，然后以低剂量（20 μg/ml）给药以维持有效性。这种Dox给药方案可以实现tTA激活基因表达的ON和OFF状态之间的快速切换。此外，妊娠大鼠给予Dox完全抑制了其后代出生后tTA激活基因的表达。母乳中存在足够水平的Dox，以在护理新生儿时产生最大功效。妊娠或哺乳期大鼠给予Dox可在胚胎和出生后发育期间持续抑制tTA依赖性基因表达。tTA转基因大鼠允许在大鼠中诱导和可逆的基因表达;这一重要工具将在人类疾病的遗传大鼠模型的开发中具有价值。
To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout time for the effects of Dox was dose-dependent. We tested a complex regime of Dox administration to determine the optimal effectiveness and washout duration. Dox was administered at a high dose (500 μg/ml in drinking water) for two days to reach the effective concentration, and then was given at a low dose (20 μg/ml) to maintain effectiveness. This regimen of Dox administration can achieve a quick switch between ON and OFF statuses of tTA-activated gene expression. In addition, administration of Dox to pregnant rats fully suppressed postnatal tTA-activated gene expression in their offspring. Sufficient levels of Dox are present in mother's milk to produce maximal efficacy in nursing neonates. Administration of Dox to pregnant or nursing rats can provide a continual suppression of tTA-dependent gene expression during embryonic and postnatal development. The tTA transgenic rat allows for inducible and reversible gene expression in the rat; this important tool will be valuable in the development of genetic rat models of human diseases.