E Cloning Of Human Sox2and Nanog Gene And Their Influences On Differentiation Of Fetal Pneumal Fibroblasts

It has been demonstrated that Homo sapiens Sox2gene and Nanog gene play a crucial role in the maintenance of the self-renewing and pluripotent state in Embryonic stem cells(ESCs). It has been the hot spot of the scientific research work.To clone Sox2and Nanog gene, and to study their molecular biology function is the focus of this study. Then sequences of target genes were ligated with carrier plasmid pSG5, copied in E. coli competent cells to get recombinant plasmids pSG5-Sox2and pSG5-Nanog, ready for the next step transfection. This experiment selected fetal pneumal interstitial fibroblasts as host cells, pSG5-Sox2and pSG5-Nanog were transfected into the fibroblasts to observe the impact of Sox2gene and Nanog gene on terminal differentiation of somatic cells.Objective1To clone the complete coding sequences (CDS) of Homo sapiens Sox2gene and Nanog gene in vitro, construct their recombinant eukaryotic expressing plasmids (pSG5-Sox2, pSG5-Nanog).2Primary culture of fetal pneumal fibroblasts as host cells and then transfecting the recombinant plasmids into the fibroblasts, to observe the expression of Sox2and Nanog and morphological changes in the fetal pneumal fibroblasts.Methods1The CDS of Sox2gene was cloned from the brain tissue of aborted fetus aged of4-6w by RT-PCR. The CDS of Nanog gene was cloned from bladder carcinoma cells. The PCR products were subjected to electrophoresis.2The obtained sequences of Sox2and Nanog were then ligated with pSG5after double-enzyme cleavage method and purification. The recombinant plasmids pSG5-Sox2and pSG5-Nanog transformed of competent cells from Escherichia coli. Colony PCR and sequence analysis were conducted for the positive clone.3Primary culture of fetal pneumal fibroblasts and passaged as host cells which divided into three groups. The experimental group’s fibroblasts were transfected by pSG5-Sox2and pSG5-Nanog with the FuGENE HD transfection reagent. Fibroblasts of the blank control group were transfected pSG5, while adding equal volume of DMEM(low glucose) with no plasmids in negative control group.4After transfecting48h, RT-PCR as well as immunocytochemical(ICC) method was carried on to detect the expression of Sox2gene and Nanog gene.15-30d after overexpression, ICC method of CK(pan), Nestin, GFAP,NSE helped to positon the certain protein in the host cells.Results1The RT-PCR products verified by electrophoresis showed complete coding sequences (CDS) of Homo sapiens Sox2gene (about lkb) and Nanog gene (about lkb). NCBI online tool BLAST illustrated sequence alignment of recombinant pSG5-Sox2, pSG5-Nanog99%similarity respectively.2The fetal pneumal fibroblasts were acquired and stably passaged. RT-PCR and ICC method revealed that pSG5-Sox2and pSG5-Nanog were expressed positively in the fetal pneumal fibroblast, while neither the blank control group nor the fetal pneumal fibroblast found no changes as mentioned above.3Before transfection, the host cells grew in palisading and whirl manner.8days after overexpression of pSG5-Sox2and pSG5-Nanog, fibroblasts in experimental group transformed into stem cell like cells.15days after overexpression, the flat, polygonal, closely packed host cells were differentiated into epithelial-like cells with positive, CK (pan) staining.20～30days after transfection, the host cells finally emerged a differentiated tendency towards neuron-like cells with obvious axon, which Nestin, GFAP and NSE staining positively. The host cells morphological changes and ICC staining of pertaining markers as mentioned above did not find in neither blank control group nor negative control group.ConclusionsIn summary, Homo sapiens Sox2gene and Nanog gene have been cloned, and the eukaryotic expressing plasmids have also been obtained in present study. Meanwhile, without feeder layer, the fetal pneumal fibroblast can differentiate into epithelial-like and neuron-like cells after days of overexpression in vitro.