Hubner Eric K, Lechler Christian, Rösner Thomas N, Kohnke-Ertel Birgit, Schmid Roland M, Ehmer Ursula
Department of Medicine II, Klinikum rechts der Isar, Technische Universität München; Department of Pneumology, Center for Medicine, Medical Center University of Freiburg.
Department of Medicine II, Klinikum rechts der Isar, Technische Universität München.
J Vis Exp. 2018 Feb 2(132):56613. doi: 10.3791/56613.
In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.
在肝癌、肝再生、炎症和纤维化的研究模型中，用于体内基因表达和沉默的灵活系统非常有用。通过尾静脉进行基于转座子构建体的流体动力学注射是对成年小鼠肝细胞进行基因操作的有效方法。除了组成型转基因表达外，该系统还可用于更先进的应用，如shRNA介导的基因敲低、应用CRISPR/Cas9系统诱导基因突变或诱导系统。在此，以组成型CreER表达与选择的转基因或miR-shRNA的诱导表达相结合为例介绍该技术。我们涵盖了从制备睡美人转座子构建体开始，到小鼠注射和处理，以及制备肝组织用于免疫染色分析的多步骤过程。所介绍的系统是在肝细胞中实现复杂基因操作的可靠且高效的方法。它与基于Cre/loxP的小鼠品系联合使用特别有用，可应用于多种肝病研究模型。
