Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2012 Aug 21;109(34):13662-7. doi: 10.1073/pnas.1211816109. Epub 2012 Aug 6.
Transgenesis promises a powerful means for assessing gene function during amphibian limb regeneration. This approach is complicated, however, by the need for embryonic appendage development to proceed unimpeded despite the genetic alterations one wishes to test later in the context of regeneration. Achieving conditional gene regulation in this amphibian has not proved to be as straightforward as in many other systems. In this report we describe a unique method for obtaining temporal control over exogenous gene expression in the axolotl. Based on technology derived from the Escherichia coli Lac operon, uninduced transgenes are kept in a repressed state by the binding of constitutively expressed Lac repressor protein (LacI) to operator sequences within the expression construct. Addition of a lactose analog, IPTG, to the swimming water of the axolotl is sufficient for the sugar to be taken up by cells, where it binds the LacI protein, thereby inducing expression of the repressed gene. We use this system to demonstrate an in vivo role for thrombospondin-4 in limb regeneration. This inducible system will allow for systematic analysis of phenotypes at defined developmental or regenerative time points. The tight regulation and robustness of gene induction combined with the simplicity of this strategy will prove invaluable for studying many aspects of axolotl biology.
转基因技术为评估在两栖动物肢体再生过程中的基因功能提供了一种强大的手段。然而，这种方法很复杂，因为需要在胚胎附肢发育不受干扰的情况下进行，尽管以后在再生的背景下需要进行基因改变的测试。在这种两栖动物中实现条件基因调控并不像在许多其他系统中那样简单。在本报告中，我们描述了一种在蝾螈中获得对外源基因表达进行时间控制的独特方法。该方法基于大肠杆菌 Lac 操纵子的技术，未诱导的转基因通过组成型表达的 Lac 阻遏蛋白（LacI）与表达构建体中的操纵序列结合而处于抑制状态。将乳糖类似物 IPTG 添加到蝾螈的游泳水中足以使细胞摄取该糖，其中它与 LacI 蛋白结合，从而诱导受抑制基因的表达。我们使用该系统证明了血小板反应蛋白-4在肢体再生中的体内作用。这种诱导型系统将允许在定义的发育或再生时间点对表型进行系统分析。基因诱导的严格调控和稳健性以及该策略的简单性将证明对研究蝾螈生物学的许多方面非常有价值。
