| Sox2 is one of the most critical genes among of sox family.Sox2 is located in chromosome 13 as a single copy and a single exon in pigs,and it acts as a transcription factor to regulate the expression of several genes,involves in development,oncogenesis and other biological events that are vital to the body.It same as OCT4 and NANOG,a core pluripotent factor,plays the important role in the mammalian early embryonic first lineage differentiation and form inner cell mass(ICM)and trophoblast(TE)two groups of different fate cell populations follow formed body and placenta,and the maintenance of pluripotency of inner cell mass.SOX2 is also a marker for various types of pluripotent stem cells and cancer cells,and an indispensable research object in the field of stem cell and regenerative medicine and cancer research.Although the character of sox2 involves in organism,the research on the regulatory network of sox2 gene is not deep,and there are few studies on its promoter regulation.It is well known that sox2 is the inverted CCAAT box element located in 468 bp upstream of the CDS can be recognized by the transcription factor NF-Y and recruits RNA polymerase to initiate transcription of sox2;positive feedback regulated by sox7 and negative by fox O1;and there are two enhancers named SRR1 and SRR2.However,the above conclusions were first derived from studies in mice,and some of the conclusions were verified in bovine(Bos taurus)and goats(Capra hircus),while there was little relevant research in pigs(Sus scrofa).The embryo is the only stable vector for expressing sox2 under the premise of no embryonic stem cell line,.According to the precious researches,there is a certain different in the pluripotent regulatory network between pig and mouse,and some conditions are closer to humans.The research in details of regulation and network of sox2 are helpful for deeply understanding the potential regulation networks and mechanisms of early embryo development.As a mammal whose physiological conditions and living habits are very similar to human beings,after understanding these contents,it will certainly provide a solid theoretical basis for its further application in human disease treatment and production practice and related basic research work.In order to systematically analyze the activity of the sox2 gene promoter in pig early embryos,the 5000 bp sequence upstream of the coding region of the pig sox2 gene was captured and predicted by the online analysis of many transcription factor binding sites,combined with the existing laboratory The transcriptome sequencing data of embryos at various stages in pigs were screened for trans-factors expressed in early embryos,and then divided into four possible "super enhancer" regions based on the distribution of binding sites of these factors.Based on this,a fragment-deleted promoter fluorescent reporter vector was designed and constructed accordingly.After the system for introducing a foreign gene into the embryo is established by means of microinjection,the promoter reporter vector to be detected is introduced into the embryo together with the internal reference plasmid.Fluorescence expression was statistically analyzed at the 4 and 8 cell stages of the embryo,and the expression level of the reporter gene was analyzed by q RT-PCR,and the activity of each promoter fragment was measured and compared.Finally,we found that in the four-cell and eightcell stages of pig embryos,the fragment from 2254 bp to 2442 bp upstream of the CDS region has strong promoter activity.In addition,we also found that the activity of a promoter fragment was detected by fluorescence analysis and q RT-PCR.There was a q RT-PCR signal but no detectable fluorescence.Since these two technical subscales represent the detection of protein levels and m RNA levels,it is concluded that there may be an unreported second transcription initiation site of the pig sox2 gene,located near the upstream of the CDS region at 1324 bp,but this conclusion remains to be Further experimental verification. |
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