Porcine Nanog Overexpression In Fetal Fibroblast And Cloned Embryos

Nanog is an important gene which plays crucial role in early embryonic development and maintaining pluripotency in ES cells. However, there is little research on Nanog in pig. Pig is considered as a useful and meaningful animal model in therapeutic and biomedical research, such as bioreactor and xenotransplantation. However, the research of these potential applications progressed slowly, because no authentic porcine ESCs are available to date. Embryonic stem cell lines provide an invaluable research tool for genetic engineering, developmental biology and disease models.These cells can be maintained indefinitely in culture and yet maintain competence to produce all the cells within a fetus. So far, validated ES cell lines have yet to be established in ungulates. The aim of this study was to establish Nanog-expressing cell lines that can be used as donor cells for nuclear transfer in pig, and further to explore Nanog function in pig. The results were listed below:(1) By reverse transcription–polymerase chain reaction (RT-PCR), the complete cDNA of Nanog gene was cloned from porcine MII stage oocytes. Confirmed by DNA sequencing, CDS of Nanog was composed of 915 nucleotides, and shared 99.8% identity with porcine Nanog in NCBI (GenBank accession no.FJ882402.1), and there are two nucleotide substitutions at 562 (G to A) and 572 (A to G) site.(2) Eukaryotic expression vectors-pEGFP-C1/Nanog and pcDNA3.1 (+)/Nanog were also successfully constructed. Both of the recombinant plasmids were transfected into fibroblast cells.Stable transfected cell lines, pEGFP-NANOG and pcDNA3.1 (+)/Nanog, were successfully established after 15-20 days of selection with neomycine (G418) and proliferation. Immunofluorescence result demonstrated that Nanog protein was successfully expressed in nucleus of porcine fetal fibroblasts (PFF).(3) We observed ES-like colonies attached to PFF after about 6-10 days G418 selection.(4) Results in the present study clearly showed ectopic expressed porcine Nanog in PFF can upregulate Oct4, C-myc and Sall4 but could not active Sox2 and Klf4. It indicates that porcine Nanog has similar properties in PFF, at least partially, which were described for Nanog in primate and murine ESCs. (5) Ectopic expressed porcine Nanog in PFF can activate LIF/STAT3 pathway and the downstream genes (Stat3 and C-myc) in PFF.(6) After culture in suspension,the pcDNA3.1(+)/Nanog expressing fibroblast cells formed typical embroid bodies,and RT-PCR detection showed that molecular markers of 3 germ layers were expressed in the embroid bodies.(7) The pluripotency assays above indicated that the Nanog-expressing cells were to some extent pluripotent(8) All reconstructed embryos employed pcDNA3.1 (+) empty vector and pcDNA3.1 (+)/Nanog vector stably transfected cells as donor cells for SCNT successfully developed to blastocyst stage but there were no statistic differences in the fusion rate, cleavage rate, and the blastocyst rate and the blastocyst cell numbers among the three groups.(9) Nanog overexpression could activate endogenous Nanog, Oct4 and Sox2 to the highest level at morula stage, the transcripts increased by 159-fold, 93-fold, and 180-fold, respectively.