Identification And Functional Analysis Of Mouse Oct4 Gene Splicing

As important biomaterials,mouse stem cells have important theoretical and practical value in embryonic development and clinical treatment.Oct4,as a core transcription factor,plays an important role in the regulation of stem cell self-renewal and pluripotency.The Oct4 transcription factor controls the development and differentiation of early embryos and is highly expressed in a variety of stem cells,including germ cells,embryonic stem cells(ESCs),embryonic germ cells(EGCs),and embryonic tumor cells(ECCs).In mouse,Oct4 plays a central role in the regulation of cellular pluripotency.By ectopic expression of transcription factors Oct4,Sox2,Klf4,and c-Myc,somatic cells can be reprogrammed into induced pluripotent stem cells(iPSCs).The human OCT4 gene can be alternatively spliced into OCT4 A,OCT4B and OCT4B1.However,studies on the function of the Oct4 splices have not been clear so far.In this study,mouse pluripotent stem cells were used as the research object,and the expression of the Oct4 gene splices was identified by different methods,and the function of the Oct4 b gene splices was analyzed.The specific results are as follows:1.Bioinformatics analysis of mouse Oct4 geneUsing bioinformatics methods for searching,comparing,and analyzing Oct4 genes,it was found that different splices of mouse Oct4 gene are located on chromosome 17,and Oct4 a and Oct4 b have the same exons 2-5.The splicing of Oct4 a and Oct4 b occurs mainly in the exon 1 and intron 1 regions,resulting in the different 5' ends.The analysis of cis-acting elements of Oct4 a and Oct4b's upstream initiation region and possible binding transcription factor showed that Oct4 a and Oct4 b may have different ways of transcriptional regulation,while the conservative analysis of Oct4 protein revealed that it is conservative of evolution between species.A transcription database search revealed that mouse Oct4 is highly expressed in germ cells and embryonic stem cells.According to reports in the literature,mouse Oct4 b may contain 4 different translation initiation sites.2.Identification of Mouse Oct4 Gene splicesUsing bioinformatics analysis,we designed several sets of specific primers to detect the expression of Oct4 and its splices in mouse pluripotent stem cells.The results showed that in addition to the reported Oct4b' and Oct4 b,the new alternative splices Oct4b1,Oct4b2 and Oct4b3 were also detected.Oct4b1 contains uncleaved intron 2 compared to Oct4 b.Oct4b2 contains an extra intron 4 compared to Oct4b1,and Oct4b3 is derived directly from the uncleaved genomic sequence.By comparing the expression patterns of the Oct4 splices in different cells,it was found that Oct4 b and Oct4 a are only expressed in pluripotent stem cells,and the expression level of Oct4 b is significantly lower than that of Oct4 a.Western blotting and immunofluorescence showed that Oct4 protein produced multiple splicing bodies in mouse pluripotent stem cells,and the expression and localization of Oct4 protein in ESCs and ECCs were different.Further studies have shown that serum concentrations in culture conditions affect the expression of Oct4 protein in ECCs.The results of different Oct4 antibodies showed that Oct4 protein includes two different cell localization and splicing modes.3.Mouse Oct4 b protein expression and localizationTo further elucidate the expression and localization of mouse Oct4 b protein,we constructed a eukaryotic expression vector containing His-tagged protein.The results showed that Oct4 b can be alternative translated to form three proteins of 246 aa,221 aa and 189 aa,while Oct4b' only forms a 189 aa protein.Oct4b2 and Oct4b3 can be alternative spliced into Oct4 b and Oct4b' to initiate the translation of proteins of the same size.Oct4 b can also be spliced into Oct4b',while Oct4b' is no longer spliced as the final splices.The Oct4 b protein has different patterns of expression in different cell types.Immunofluorescence staining revealed that the Oct4 b protein is mainly located in nucleus.4.Functional analysis of mouse Oct4 gene splicesThe internal ribosome insertion site(IRES)element at the 5' end of the Oct4 b sequence not only alternative initiates translation to form different Oct4 b protein,it also has promoter activity.By transfecting different model cells,the results showed that there is a difference in the translational expression pattern of the mouse Oct4 b IRES element in pluripotent and non-pluripotent cell lines.By comparing the expression changes of Oct4 b splices when different pluripotent cells were differentiated,the results showed that the expression level of Oct4 b splices was decreased significantly with the decrease of Oct4 a expression.When the pluripotency of miPS cells was slightly decreased,Oct4 a expression and AP staining could not accurately show the difference in cellular pluripotency,but Oct4b' could serve as a more accurate pluripotency marker gene.