Reprogramming Of Amur Tiger Fibroblasts To Induced Pluripotent Stem Cells Using Oct-4、Sox2、Klf4and C-Myc Transcription Factors

Biodiversity protection, which includes the rescue and protection of endangered species, has become an important mission all over the world. Tigers are one of the wild flagship species that have been receiving a lot of attention. The Amur tiger(Panthera tigris altaica), honored as the king of the forest, is at the top of the food chain in the forest ecosystem and plays a critical role in the natural ecosystem of the forests they inhabit. However, direct hunting by humans has destroyed the environment in which they survive and, as a result, the population of the wild Amur tiger has dropped markedly. Currently, Amur tigers live only in the Far East regions of Russia and in Northeast China. The application of cellular and molecular biotechnology techniques to studying the genetic resources of the Amur tiger will be essential in restoring the wild habitats of the Amur tiger and in protecting the Amur tigers that still exist. In this study, various cellular and molecular biotechnologies were used to investigate the following aspects that are important in conserving the genetic resources of the Amur tiger:1. Fibroblast cell lines of Amur tiger were established using adherent cell culture and cryobiology. The cell line quality and biological features indicated that:the fibroblasts cultured in vitro grew well and the morphology was the typical long spindle shape; the growth cycle after cryopreservation and resuscitation included a latent stage, an exponential growth stage, and a plateau stage; and the growth curve was the typical "S"-shape with a doubling time of30h. A microbiological examination was negative in bacteria, fungi, virus, and mycoplasma, and the isozyme zymogram of lactate dehydrogenase and malate dehydrogenase was species-specific without inter-species contamination. The diploid cell rate was97.83%, and the expression rates of six fluorescent protein genes were9.81-23.19%. All indices of fibroblasts of the Amur tiger met the cellular identification standard of the American Type Culture Collection (ATCC).2. The Amur tiger genomic DNA was extracted using low-melting agarose. We constructed a BAC vector with T4DNA ligase and used electrotransfection to build a BAC library that had153600BAC clones and6.5-fold coverage. The average length of insertions was116.5kb with a blank clone rate of2.6%. The BAC library of Amur tiger was used to construct the subsequent functional gene clone library and physical spectrum.3. We used high throughput de novo transcriptomic sequencing of total RNA in the lung and placenta tissues of Amur tiger for sequencing analysis. We obtained9.2GB of data and detected80624genes that were548-575bp in length. The sequences of48.3%of these genes were annotated by homology searches against the Nr, Swiss-Prot, KEGG, and COG databases. We found that a large number of the predicted functional genes of Amur tiger were highly homologous to genes of domestic cats. We then used GO and Pathway analysis to relate some of the functional genes in specific tissues to networks. In addition, we obtained the mRNA sequences of the pluripotent genes of Oct4, Sox2, Klf4, and c-Myc mRNA, which provided a theoretical basis for the reported cellular re-programming.4. We cloned the pluripotent genes of Oct4Sox2, K1f4, and c-Myc by PCR in vitro, and created an over-expression lentiviral vector by linking the genes and the lentiviral vector of Lv-efla-eGFP-tre-X. We then transfected293T cells using liposome2000after extracting the plasmid for packaging, collection, and concentration, and obtained a high viral titer of1.1×108TU/ml.5. We transformed the Oct4, Sox2, Klf4, and c-Myc Amur tiger genes to the Amur tiger fibroblasts to get stable expression, and obtained iPS clones after14days of induction. The surface markers of the stem cells (Oct-4, Nanog, Sox2, SSEA-1, SSEA-3, SSEA-4, Tra-1-60, Tra-1-80) were verified by immunofluorescence and western blot. A karyotype analysis indicated that there was no genetic variation of the iPS cells during re-programming. We used real-time PCR to analyze the expressions of Oct-4, Sox2, Nanog, TRET, Bax, Bcl-xl, and P53in the Amur tiger iPS cells during re-programming. Alkaline phosphatase staining showed high expression of alkaline phosphatase in the iPS cells, the differentiation ability of the iPS cells was confirmed by injection of teratoma formed in immunodeficient mice. The in vitro directional induced differentiation of three blastoderms confirmed the in vitro differentiation of the Amur tiger iPS cells.In summary, in the present study we obtained the gene sequences of Oct4, Sox2, K1f4, and c-Myc of Amur tiger using high-throughput sequencing to construct a lentiviral vector that was expressed stably in fibroblasts, and induced the fibroblasts to be re-programmed to iPS cells that met the standard of pluripotent stem cells. The successful establishment of Amur tiger iPS cell lines not only provides a cell model for the early embryonic development of the Amur tiger but also makes available a model that can be stored and used as a genetic resource for a long time. Therefore, there was first time to realize the protection of Amur tiger in our country via three pathway, which including Ex-situ conservation, In-situ conservation and In vitro conservation.