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| Effect of Overexpression of SET and MYND Domain Containing Protein 3 Gene (SMYD3) in Bovine (Bos taurus) Embryonic Fibroblast Cells (bEFs) on the Cells Proliferation and the Efficiency for Induced Pluripotent Stem Cells (iPSCs) |
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Abstract SET and MYND domain-containing protein 3 (SMYD3) is a histone H3 Lysine 4 trimethyltransferase, which overexpresses in many cancers and can promote cells proliferation, migration and invasion. In order to study the function of SMYD3 in bovine embryonic fibroblast cells (bEFs), SMYD3 CDS was obtained from bovine (Bos taurus) and inserted into the vector pIRES2-Zsgreen1 to construct overexpression vector pSMYD3-IRES2-Zsgreen1. pSMYD3-IRES2-Zsgreen1 was transfected into bEFs and qRT-PCR and Western blot were used to verify its expression. Also, the cell growth was recorded by detecting the absorbancy of 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di- phenytetrazoliumromide (MTT) every 24 h for 7 d. At last, the transgenic bEFs were infected with lentiviral vectors expressing the mouse (Mus musculus) octamer-binding transcription factor 4 (Oct4), SRY (sex determining region Y)-box 2 (Sox2), avian myelocytomatosis viral oncogene homolog (c-Myc) and Kruppel-like factor 4 (Klf4) transcription factors, respectively, to obtain the bovine induced pluripotent stem cells (iPSCs). The enzyme digestion results showed that the vector pSMYD3-IRES2-Zsgreen1 was correctly constructed. The qRT-PCR and Western blot results indicated that SMYD3 overexpressed in pSMYD3-IRES2-Zsgreen1 transfected bEFs, which cells growth improved comparing with the control vector transgenic cells. After induction by the 4 transcription factors, the SMYD3 transgenic cells formed more stem cell-like clones, which displayed OCT4, NANOG homeobox (NANOG) and SOX2 immunofluorescence staining positively. These results established and firstly reported that SMYD3 could promote bEFs growth and induction efficiency for iPSCs, which provides basic data for the strategies of preparing bovine iPSCs and improves understanding of the molecular basis of iPSCs.
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Received: 24 March 2015
Published: 24 July 2015
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