Establishment Of Bactrian Camel Transgenic Cells Mediated By CRISPR/Cas9 And Its Exploration For IPS Cell Induction

Bactrian camels are endemic species in the northwestern region of China.Due to environmental and other factors,the number of wild bactrian camels is rapidly decreasing.It have great significance for choosing suitable methods to protect its germplasm resources.Considering that bactrian camel is more special physiology,it is feasible to use the induced pluripotent stem cells to protect the genetic resources of bactrian camel.CRISPR/Cas9 technology is the latest gene editing technology.The CRISPR/Cas9 full name is clustered regularly interspaced short palindromic repeats/Cas9.In theory,gene cutting can be performed at any position in the whole genome and confirmed in multiple species.Combining the above two more mature technologies,the Bactrian camel transgenic cells were successfully constructed in this experiment,providing biomaterials for the optimization of screening iPS cells in the later period,and the conditions for inducing iPS cells were preliminary explored.The main results are as follows:1.Construction of expression vectors According to the phenomenon that the Nanog gene is only expressed in pluripotent cells,upstream and downstream homologous arms are designed,and the lengths are 2341 bp and 1775 bp,respectively.At the same time,the eGFP gene and the neo gene were cloned.In this experiment,the upstream and downstream homologous arms were first ligated into pUC,then the neo gene with a eukaryotic promoter was ligated,and finally the eGFP gene was ligated to successfully obtain the expression vector pUC-up-eGFP-pgk-down.2.Construction of the cleavage vector Using online software,three pairs of sgRNAs were designed near the stop codon of the Nanog gene according to the principle of 20+NGG.It was ligated into pX330-eGFP.3.Bactrian camel transgenic cells were obtained The three pairs of cleavage vectors were transfected into Bactrian camel fetal fibroblasts,and the respective cleavage efficiency was detected by T7E1 digestion.The Bactrian camel fibroblasts were co-transfected with the cutting vector with the highest cutting efficiency and the expression vector.Through G418 selection,cells with successful knock-in were obtained and used for subsequent i PS cell induction.4.Exploration on Condition of iPS Cell Induction in Transgenic Cells of Bactrian Camel Four genes,Oct4,Sox2,Klf4,and c-Myc,were transfected into Bactrian camel transgenic cells using retroviruses,and induced by different culture protocols in a hypoxic environment.Alkaline phosphatase assay was performed after 16 days,indicating that the induction conditions need to be optimizedIn summary,two genes,the eGFP gene and the neo gene with a eukaryotic promoter,were knocked in at a fixed point after the Nanog gene in fetal buffalo fetal fibroblasts.Bactrian camel transgenic cells were successfully constructed,and biomaterials were provided for the optimization of screening iPS cells in the later period,and the conditions for inducing iPS cells were preliminary explored.