a virus-free poly-promoter vector induces pluripotency in quiescent bovine cells under chemically defined conditions of dual kinase inhibitionpoly-promoter无毒化向量诱发化学定义条件下静止牛细胞多能性的双重激酶抑制.pdfVIP

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a virus-free poly-promoter vector induces pluripotency in quiescent bovine cells under chemically defined conditions of dual kinase inhibitionpoly-promoter无毒化向量诱发化学定义条件下静止牛细胞多能性的双重激酶抑制.pdf

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a virus-free poly-promoter vector induces pluripotency in quiescent bovine cells under chemically defined conditions of dual kinase inhibitionpoly-promoter无毒化向量诱发化学定义条件下静止牛细胞多能性的双重激酶抑制

A Virus-Free Poly-Promoter Vector Induces Pluripotency in Quiescent Bovine Cells under Chemically Defined Conditions of Dual Kinase Inhibition 1. 1,2. 3 4 1 1 Ben Huang , Tong Li , Lucia Alonso-Gonzalez , Ruben Gorre , Sarah Keatley , Andria Green , Pavla 1 1 5 ¨ 1 Turner , Prasanna Kumar Kallingappa , Vinod Verma , Bjorn Oback * 1 AgResearch, Ruakura Research Centre, Hamilton, New Zealand, 2 Animal Reproduction Institute, Guangxi University, Nanning, China, 3 Children’s Cancer Research Group, Department of Paediatrics, University of Otago, Christchurch, New Zealand, 4 Trenzyme GmbH, Konstanz, Germany, 5 Department of Biochemistry, National University of Singapore, Singapore, Singapore Abstract Authentic induced pluripotent stem cells (iPSCs), capable of giving rise to all cell types of an adult animal, are currently only available in mouse. Here, we report the first generation of bovine iPSC-like cells following transfection with a novel virus- free poly-promoter vector. This vector contains the bovine cDNAs for OCT4, SOX2, KLF4 and c-MYC, each controlled by its own independent promoter. Bovine fibroblasts were cultured without feeders in a chemically defined medium containing leukaemia inhibitory factor (LIF) and inhibitors of MEK1/2 and glycogen synthase kinase-3 signaling (‘2i’). Non-invasive real- time kinetic profiling revealed a different response of bovine vs human and mouse cells to culture in 2i/LIF. In bovine, 2i was necessary and sufficient to induce the appearance of tightly packed alkaline phosphatase-positive iPSC-like colonies. These colonies formed in the absence of DNA synthe