An in vivo transfection system for inducible gene expression and gene silencing in murine hepatocytes.
An in vivo transfection system for inducible gene expression and gene silencing in murine hepatocytes.
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用于小鼠肝细胞中诱导基因表达和基因沉默的体内转染系统。
DOI：
10.1002/jgm.2940
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Ehmer,Ursula
中科院分区：
文献类型：
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作者：
Hubner,EricK;Lechler,Christian;Kohnke-Ertel,Birgit;Zmoos,Anne-Flore;Sage,Julien;Schmid,RolandM;Ehmer,Ursula
背景转座子整合载体的流体动力学尾静脉注射（HTVI）是一种稳定的体内转染小鼠肝细胞的系统，已成功用于研究肝脏生物学和癌症的关键问题。改进转座子介导的肝细胞转染载体将进一步扩大该技术在肝脏研究中的应用范围。在本研究中，我们报告了一个先进的转座子为基础的系统，用于操纵基因表达在hepatocytesinvivo.MethodsTransposon为基础的载体构建体，使组成型表达的诱导型Cre重组酶（CreER）与四环素诱导转基因或miR-小发夹RNA（shRNA）表达（泰特-ON系统）。通过HTVI将转座子和转座酶表达载体共注射到R26 R-mTmG报告基因小鼠中。Cre介导的基因重组由他莫昔芬诱导，然后给予多西环素以驱动四环素诱导的基因或shRNA表达。通过免疫荧光染色在注射mice. ResultsHTVI后，Cre重组他莫昔芬导致膜结合的绿色荧光蛋白在转染的肝细胞中的表达可视化的表达。诱导基因或shRNA表达的激活通过免疫染色在多达三分之一的转染肝细胞中检测到，其效率依赖于驱动泰特-ON system.ConclusionsOur载体系统的启动子结合Cre‐lox介导的基因突变与诱导基因表达或基因敲低，分别。它为肝细胞基因表达的快速和特异性修饰提供了机会，并且可以成为遗传筛选方法和特异性地在基因工程小鼠模型中分析靶基因的有用工具。
BackgroundHydrodynamic tail vein injection (HTVI) of transposon‐based integration vectors is an established system for stably transfecting mouse hepatocytesin vivothat has been successfully employed to study key questions in liver biology and cancer. Refining the vectors for transposon‐mediated hepatocyte transfection will further expand the range of applications of this technique in liver research. In the present study, we report an advanced transposon‐based system for manipulating gene expression in hepatocytesin vivo.MethodsTransposon‐based vector constructs were generated to enable the constitutive expression of inducible Cre recombinase (CreER) together with tetracycline‐inducible transgene or miR‐small hairpin RNA (shRNA) expression (Tet‐ON system). Transposon and transposase expression vectors were co‐injected intoR26R‐mTmGreporter mice by HTVI. Cre‐mediated gene recombination was induced by tamoxifen, followed by the administration of doxycycline to drive tetracycline‐inducible gene or shRNA expression. Expression was visualized by immunofluorescence staining in livers of injected mice.ResultsAfter HTVI, Cre recombination by tamoxifen led to the expression of membrane‐bound green fluorescent protein in transfected hepatocytes. Activation of inducible gene or shRNA expression was detected by immunostaining in up to one‐third of transfected hepatocytes, with an efficiency dependent on the promoter driving the Tet‐ON system.ConclusionsOur vector system combines Cre‐lox mediated gene mutation with inducible gene expression or gene knockdown, respectively. It provides the opportunity for rapid and specific modification of hepatocyte gene expression and can be a useful tool for genetic screening approaches and analysis of target genes specifically in genetically engineered mouse models.