Abbey Deepti, Singh Gurbind, Verma Isha, Derebail Suchitra, Kolkundkar Udaykumar, Chandrashekar Darshan S, Acharya Kshitish K, Vemuri Mohan C, Seshagiri Polani B
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.
Present address: Centre for Stem Cell Research, Christian Medical College Campus, Bagayam, Vellore, India.
Cell Reprogram. 2019 Oct;21(5):270-284. doi: 10.1089/cell.2019.0019.
The embryonic stem cell line derivation from nonpermissive mouse strains is a challenging and highly inefficient process. The cellular reprogramming strategy provides an alternative route for generating pluripotent stem cell (PSC) lines from such strains. In this study, we successfully derived an enhanced green fluorescent protein (EGFP)-transgenic "N9" induced pluripotent stem cell (iPS cell, iPSC) line from the FVB/N strain-derived mouse embryonic fibroblasts (MEFs). The exposure of MEFs to human , , , and (OSKM) transgenes via lentiviral transduction resulted in complete reprogramming. The N9 iPS cell line demonstrated all the criteria of a typical mouse PSC line, including normal colony morphology and karyotype (40,XY), high replication and propagation efficiencies, expression of the pluripotency-associated genes, spontaneous differentiation to three germ lineage-derived cell types, and robust potential of chimeric blastocyst formation. Taken together, using human OSKM genes for transduction, we report, for the first time, the successful derivation of an EGFP-expressing iPS cell line from a genetically nonpermissive transgenic FVB/N mouse. This cell line could provide opportunities for designing protocols for efficient derivation of PSC lines from other nonpermissive strains and developing mouse models of human diseases.
从不允许的小鼠品系中获得胚胎干细胞系是一个具有挑战性且效率极低的过程。细胞重编程策略为从这些品系中产生多能干细胞（PSC）系提供了一条替代途径。在本研究中，我们成功地从FVB/N品系来源的小鼠胚胎成纤维细胞（MEF）中获得了一种增强型绿色荧光蛋白（EGFP）转基因的“N9”诱导多能干细胞（iPS细胞，iPSC）系。通过慢病毒转导使MEF暴露于人源的、、、和（OSKM）转基因，导致完全重编程。N9 iPS细胞系展示了典型小鼠PSC系的所有标准，包括正常的集落形态和核型（40,XY）、高复制和增殖效率、多能性相关基因的表达、自发分化为三种胚层来源的细胞类型以及强大的嵌合囊胚形成潜力。综上所述，利用人源OSKM基因进行转导，我们首次报道了从基因上不允许的转基因FVB/N小鼠成功获得表达EGFP的iPS细胞系。该细胞系可为设计从其他不允许的品系中高效获得PSC系的方案以及开发人类疾病的小鼠模型提供机会。
