Pieters Tim, Haenebalcke Lieven, Bruneel Kenneth, Vandamme Niels, Hochepied Tino, van Hengel Jolanda, Wirth Dagmar, Berx Geert, Haigh Jody J, van Roy Frans, Goossens Steven
Department of Biomedical Molecular Biology, Ghent University; Inflammation Research Center, VIB; Center for Medical Genetics, Ghent University Hospital; Cancer Research Institute Ghent (CRIG);
Department of Biomedical Molecular Biology, Ghent University; Inflammation Research Center, VIB.
J Vis Exp. 2017 Apr 27(122):55575. doi: 10.3791/55575.
Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.
对小鼠胚胎或胚胎干细胞（mESCs）进行基因工程操作有助于研究特定蛋白质的功能。蛋白质是细胞的“主力军”，通常由多个功能域组成，这些功能域会受到翻译后修饰的影响。在条件性或组成型基因敲除（KO）小鼠中使整个蛋白质缺失，并未考虑到这种功能多样性和调控。此前报道了一种mESC系和一种衍生的小鼠模型，其中在ROSA26（R26）位点内插入了用于FLPe重组介导的盒式交换（RMCE）的对接位点。在此，我们报道一种结构-功能研究方法，该方法能够对多结构域蛋白质的不同功能进行分子剖析。为此，必须将RMCE兼容小鼠与KO小鼠杂交，然后分离出RMCE兼容的KO mESCs。接下来，可以通过RMCE靶向将一组推定的拯救构建体引入R26位点。使用重组克隆可将候选拯救cDNA轻松插入靶向载体的RMCE位点之间。接下来，将靶向载体与FLPe重组酶表达质粒一起转染KO mESCs。RMCE可激活ROSA26对接位点中无启动子的新霉素抗性基因，并允许选择正确的靶向事件。通过这种方式，可获得接近100%的高靶向效率，从而能够以半高通量方式插入多个推定的拯救构建体。最后，可以测试大量由R26驱动的拯救构建体拯救在亲本KO mESCs中观察到的表型的能力。我们使用胚状体（EBs）中的内胚层分化作为表型读数，在p120连环蛋白（p120ctn）KO mESCs中进行了原理验证结构-功能研究。这种方法能够识别给定蛋白质KO表型背后的重要结构域、推定的下游途径以及与疾病相关的点突变。
