Using Zinc Finger Nuclease Technology to Generate CRX-Reporter Human Embryonic Stem Cells as a Tool to Identify and Study the Emergence of Photoreceptors Precursors During Pluripotent Stem Cell Differentiation.
Using Zinc Finger Nuclease Technology to Generate CRX-Reporter Human Embryonic Stem Cells as a Tool to Identify and Study the Emergence of Photoreceptors Precursors During Pluripotent Stem Cell Differentiation.
复制标题
DOI：
10.1002/stem.2240
复制
发表时间：
2016-02
期刊：
影响因子：
--
通讯作者：
Lako M
中科院分区：
文献类型：
--
作者：
Collin J;Mellough CB;Dorgau B;Przyborski S;Moreno-Gimeno I;Lako M
本研究的目的是产生在视网膜发育中的关键转录因子锥杆同源盒（CRX）基因的内源基因座处携带绿色荧光蛋白（GFP）报告基因的人胚胎干细胞（hESC）系。将设计用于在CRX的3′ UTR中切割的锌指核酸酶（ZFN）与含有与靶区域同源性的供体构建体、eGFP报告基因和嘌呤霉素选择盒一起沿着转染到hESC中。选择后，抗生素耐药克隆的聚合酶链反应（PCR）和测序分析表明报告盒在CRX基因的3′端靶向整合，产生CRX-GFP融合体。对表现出GFP报告基因的纯合子整合的克隆进行进一步分析，表明基因组稳定性得到保留，并且没有靶向盒的其他拷贝插入基因组内的其他地方。选择该克隆用于向视网膜谱系分化。免疫细胞化学的部分获得的胚状体和定量逆转录酶PCR的GFP阳性和阴性亚群纯化的荧光激活细胞分选在分化过程中表明GFP和内源性CRX表达之间的显着相关性。此外，GFP的表达被发现在hESC分化过程中出现的感光细胞前体，但不是在视网膜色素上皮，视网膜神经节细胞，或发育中的内核层的神经元。总之，我们的数据证明了ZFN技术成功应用于产生CRX-GFP标记的hESC系，其可用于在hESC分化期间研究和分离感光细胞前体。干细胞2016;34：311-321
The purpose of this study was to generate human embryonic stem cell (hESC) lines harboring the green fluorescent protein (GFP) reporter at the endogenous loci of the Cone‐Rod Homeobox (CRX) gene, a key transcription factor in retinal development. Zinc finger nucleases (ZFNs) designed to cleave in the 3′ UTR of CRX were transfected into hESCs along with a donor construct containing homology to the target region, eGFP reporter, and a puromycin selection cassette. Following selection, polymerase chain reaction (PCR) and sequencing analysis of antibiotic resistant clones indicated targeted integration of the reporter cassette at the 3′ of the CRX gene, generating a CRX‐GFP fusion. Further analysis of a clone exhibiting homozygote integration of the GFP reporter was conducted suggesting genomic stability was preserved and no other copies of the targeting cassette were inserted elsewhere within the genome. This clone was selected for differentiation towards the retinal lineage. Immunocytochemistry of sections obtained from embryoid bodies and quantitative reverse transcriptase PCR of GFP positive and negative subpopulations purified by fluorescence activated cell sorting during the differentiation indicated a significant correlation between GFP and endogenous CRX expression. Furthermore, GFP expression was found in photoreceptor precursors emerging during hESC differentiation, but not in the retinal pigmented epithelium, retinal ganglion cells, or neurons of the developing inner nuclear layer. Together our data demonstrate the successful application of ZFN technology to generate CRX‐GFP labeled hESC lines, which can be used to study and isolate photoreceptor precursors during hESC differentiation. Stem Cells 2016;34:311–321