Synchronously Transgene of Therapeutic Intercalated Disc-related CX43 and ZO1 Genes and Reprogramming Genes does not Affect The Pluripotent of Pig IPS

Abstract

AM: To explore the feasibility of reprogramming pig somatic cells into inducible pluripotent stem cells carrying therapeutic intercalated disc-related CX43 single gene and CX43/ZO1 dual genes. METHODS: The retroviral vectors of CX43, ZO1, Oct-4, Sox2-HUMAN, Nanog, and Lin28 were synchronously transfected into the pig dermal fibroblasts to construct three iPS cell lines: iPS cell, iPS cell carring CX43 single gene and iPS cell carring CX43/ZO1 dual genes. The iPS cells’ surface markers including SSEA-3, SSEA-4, nanog, REX-1, TRA-1-60, and TRA-1-81 were detected by immunohistochemical staining. In-vitro EB formation and in-vivo teratoma formation were done to observing the pluripotent differentiation abilities of iPS cells. RESULTS: Four~five weeks after transduction, colonies resembling ES cells of three iPS lines emerged. Cells of three iPS cell lines all expressed REX-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. All of the three iPS cell lines showed alkaline phosphatase activity. Suspension cultures of the three iPS cell lines vitnessed Ebs formation. After injecting cells of each iPS cell line into the back of NOD/shi-scid and IL-2Rγnull (NOG) mice, teratomas formed, including ectodermal, mesodermal, and endodermal tissues. CONCLUSIONS: Reprogramming pig iPS cells carrying CX43 single gene or CX43/ZO1 dual genes is feasiblity, and synchronously transgene of therapeutic intercalated disc-related CX43 and ZO1 genes and reprogramming genes does not affect the pluripotent of pig iPS.