Establishment And Identification Of Bactrian Camel Induced Pluripotent Stem Cells And Screening Of Unique Induced Genes

Bactrian camel is a unique animal species in desert and semi-desert areas.It is resistant to high temperature,high cold,salt and alkali,and has a long reproductive cycle.It is a "multi-purpose livestock" with a special reproductive physiological structure.It also plays an important role in medical,health care,immunology and other fields,and the wild Bactrian camel is the eighth most endangered species.Therefore,it is very important to study the hereditary characters of Bactrian camels and protect their germplasm resources.Poorly differentiated stem cells have great advantages in this regard,but the isolation of large livestock and cloven-hoofed embryonic stem cells(ESCs)has been difficult to achieve,so induced pluripotent stem cells(i PSCs)have become the best choice.So far,i PSCs have been successfully generated from various animals including humans,mice,sheep,cattle,rats,rabbits,pigs,horses and monkeys.But there is no report on Bactrian camel induced pluripotent stem cells(bci PSCs).In this study,the target cells were obtained by primary culture.Referring to the induction scheme of i PSCs of other species,the retrovirus packaging system was used to introduce foreign genes,and the clones of Bactrian camel i PSCs were obtained and passaged.The detection was performed by alkaline phosphatase staining(Alkaline phosphatase,AP),pluripotency-related gene detection,immunofluorescence experiment,methylation detection,embryoid body experiment and teratoma experiment.It was proved that the Bactrian camel i PSCs cell line was successfully obtained.The specific results are as follows:1.Bactrian camel fetal fibroblasts(BCFFs)were successfully separated and obtained,and their purity and the presence or absence of mycoplasma contamination were detected,and the requirements for subsequent reprogramming experiments were achieved through detection;mouse embryonic fibroblasts(MEFs)were successfully isolated and obtained,and no mycoplasma contamination was detected;p MXS-EGFP plasmid was successfully constructed,and the corresponding infectious pseudovirus was packaged.2.To explore the mechanism of valproic acid(VPA)in the early stage of reprogramming of Bactrian camel BCFFs,and preliminarily determined that: on the one hand,it inhibits the cell cycle signaling pathway,so that all cells stay in the G2/M phase,and significantly increase Reprogram the expression of the endogenous gene c-Myc,so that the reprogrammed cells break the restriction of staying in the G2/M phase and directly improve the reprogramming efficiency;on the other hand,VPA can significantly increase the expression of VEGFC and other genes,to promote the transformation of fibroblasts to endothelial cells(this is different from the process of fibroblast-to-thelial transformation of other species).3.The Bactrian camel i PSCs cell line was successfully constructed: after identification,the AP detection in the obtained cell line was positive;except for the Klf4 gene,the other three exogenous genes were expressed to varying degrees;n Sox2,n Nanog and other endogenous genes were activated;Nanog,Oct4,Sox2,SSEA1,SSEA3,SSEA4,Rex1,Tra-1-60 and Tra-1-81 were all positive in bci PSCs;telomerase(TERT)activity was significantly increased;Nanog gene promoter region had different degrees of demethylation;karyotype was normal,diploid;embryoid body test proved that it has the ability to differentiate into three germ layer cells in vitro;The teratoma test proved that it has the ability to differentiate into three germ layers cells or tissues in vivo;RNA-Seq sequencing results showed that the Bactrian camel i PSCs transcriptome significantly differential gene expression trend is closest to that of human ESCs,and more similar to porcine i PSCs;the change trends of pluripotency-related genes,important signaling pathway genes and methylation-related genes were also very similar to those of human ESCs.4.Transcriptional data of bactrian camel i PS cells were screened by direct analysis(significantly enriched GO term and signal pathway)and known induction gene analysis,there were 46 core genes were obtained.By screening the differentially up-regulated genes in the omics data,42 complementary genes were obtained,and only 3 genes were shared with the core genes.A total of 85 candidate specific induction genes of BACtrian camel i PS cells were obtained.In this study,Bactrian camel i PSCs with differentiation potential were obtained and unique candidate induced genes were screened.It provides a theoretical basis for the subsequent improvement of the Bactrian camel i PSCs induction method and the establishment of Bactrian camel embryonic stem cells,and provides materials for Bactrian camel functional gene research,genetic characteristics research and germplasm resource protection.