Analyzing Bovine OCT4 And NANOG Enhancer Activity In Pluripotent Stem Cells Using Fluorescent Protein Reporters

Induced pluripotent stem(iPS)cells are invaluable tools for embryo development study,generation of bioreactor,new production trait and disease models.Low efficiency is always an obstacle cell reprogramming research,especially in domestic animal area.Since the establishement of transgenic mice which carry pluripotent gene green fluorescent protein reporter has,more and more pluripotent gene reporter are applied in cell reprogramming studies.Green fluorescent protein(GFP)reporters controlled by the regulatory region of OCT4 and NANOG-two master regulators for pluripotency are widely used in studies of pluripotent stem cell establishment and embryo development.Alongside the challenge in establishing bovine pluripotent stem cells,the application of bovine-specific gene reporters has rarely been explored.Using lentivirus-based GFP reporter,we investigated the upstream regulatory regions of bovine OCT4 and NANOG.The lentiviral-based fluorescent reporters controlled by bovine OCT4 and NANOG will serve as versatile tools for bovine ESC/iPSC generation and identification,as well as for developmental studies of bovine embryos.The results are shown as below:(i)Using pGreenFire lentivirus-based vector,we generated bovine specific OCT4 and NANOG enhancer-controlled lentiviral GFP reporters.Bovine genomic DNA extracted from bovine Wharton's jelly cells.OCT4 and NANOG enhancer was amplified and inserted into vector to produce virus.Mouse and human embryo stem cells were infected by the reporter viruses.The mouse and human embryo stem cells infected by these reporter viruses were found expressing GFP under the microscope.At last,we evaluated if these reporters can be silenced after the infected mouse embryo stem cells were differentiated into embryo bodies.Using flow cytometry detecting cells derived from the embryo bodies,the green fluorescence of cells was found decreased.Meanwhile the OCT4 expression level in embryo body cells,through real time qPCR measurement,was decreased too.We found the intensity of the green fluorescence was correlated with the OCT4 expression level during the cell differentiation.This result indicated these reporters show activity in both naive-and primed-state pluripotency when infected into mouse and human embryonic stem cells(ESCs),respectively.And the activity of bOCT4-GFP reporter does decrease upon the differentiation of these ESCs.In general,bovine OCT4 and NANOG reporter can show activity in both Naive and Prime state pluripotent cells and they can be used to reflect the stem cell pluripotence.This reporter system can make a preparation for detailed bovine OCT4 enchancer analyzing.(2)OCT4 expression was regulated by its distal and proximal enchancar in pluripotent cells.Using pGreenFire lentivirus-based vector,we generated bovine specific OCT4 distal and proximal enhancer-controlled lentiviral GFP reporter bOCT4-DE-GFP,bOCT4-DE2-GFP and bOCT4-PE-GFP.Bovine OCT4 distal and proximal enhancer was cloned into pGreenFire vector using bovine Wharton's jelly cells genomic DNA.After packaging these construct into lenti-virus,we infected these virus into mouse embryo stem cells.Consistent with what is found in humans and mice,the bovine OCT4-distal enhancer(bOCT4-DE)but not the proximal enhancer(bOCT4-PE)region is preferentially activated in naive-state pluripotency.Furthermore,the bOCT4-DE region is silenced upon conversion of Naive-state ESCs into primed-state epiblast stem cells(EpiSCs).In the end,we checked these reporters can be silenced when the infected mouse embryo stem cells were differentiated into embryo bodies.This resulted indicated that bovine OCT4 distal enhancer only show activity in naive state stem cells and silenced upon the differentiation of ESCs.These OCT4 distal and proximal enhancer-controlled lentiviral GFP reporters can give a good help for bovine Naive state ECS isolation,culture and bovine Naive state iPSCs study.(3)To test if the bOCT4-GFP reporters we generated could be used as live indicators in reprogramming cells for iPSC generation.We co-infected mouse fibroblasts with the reprograming factors for induced pluripotent stem cell(iPSC)induction,which leads to the generation of GFP positive colonies.Removing the doxycycline(Dox),which inducing the reprogramming factors expression,and transfer the iPS colonies into 2i/Lif medium which is a restricted condition for naive state stem cell culture,the colonies can grow healthily and contitue expressing GFP.Examing these clonies using Real-time qPCR shows that the pluripotent gene expressed in induced cells can reached into stem cell levels.That means the colonies has been completed reprogrammed and the colonies still expressed green fluorescence,indicated that the fluorescence was induced by pluripotency circuitry genes.These results demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment.In summary,we generated bovine specific OCT4 and NANOG enhancer-controlled lentiviral GFP reporters.These reporters show activity in mouse and human ESCs,with reporters regulated by bovine OCT4-DE region activated preferentially in naive-but not primed-state pluripotent cells.The OCT4-DE region is silenced upon conversion from naive-to primed-state,and can be reactivated when converted back from primed-to naive-state pluripotency.Co-infection of mouse embryonic fibroblasts with the OKSM factors for iPSC induction leads to the generation of GFP+ colonies,demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment.These reporters will serve as useful tools for completely reprogrammed bovine ESC/iPSC generation,as well as for early bovine embryo development studies.