| Multiple transcription factors such as Oct4,Sox2,Klf4 and c-Myc are introduced into mature somatic cells and reprogrammed to produce pluripotent cells called induced pluripotent stem cells(iPSCs).iPSCs can differentiate into all cell types in the body and have great potential for application in medicine,agriculture and biotechnology.However,the molecular mechanisms behind reprogramming remain largely unknown and have prevented the creation of true iPSCs and their use in clinical practice.Based on this study,cultivation of rabbit fetal fibroblasts in the cloning of specific transcription factors and slow virus carrier to build,the rabbit fetal fibroblasts induced by experimental research,such as iPSCs on rabbit iPSCs induction system and build a more perfect,more efficient rabbit somatic cell reprogramming to explore the method of iPSCs,iPSCs for animal husbandry and the application of life science to provide the reference.(1)Isolation and culture of rabbit fibroblasts and preparation of feeding layer.Rabbit fetal fibroblasts of 14 to 16 days of gestational age were successfully isolated by trypsin digestion method in vitro.The isolated cells were inoculated in culture flask,and REF grew adherently after 3 to 4h.2D cells reached 80%to 90%fusion degree,and after 2 to 3 times of passage,epithelial cells and other hybrid cells were removed to obtain high purity REF.The shape of the REF obtained was typical,with long spindle shape and growth curve close to "S" shape.Ref of p3-p5 generation with high purity and good activity was selected to make feeder layer.After being treated with mitomycin C for 2.5 hours,REF showed no obvious changes in cell morphology and good cell viability,which could be used for the subsequent culture of iPSCs.Ref with typical morphology,high purity and good growth state were successfully isolated and cultured in the experiment.By observing its growth characteristics,the rules of its in vitro culture were summarized,so as to provide sufficient and reliable seed cells for the study of iPSCs.(2)Cloning of iPSCs-related specific transcription factors and construction of lentiviral vector.According to the rabbit Oct4,Sox2,Klf4,c-Myc,Naong and Lin28 sequence information published in NCBI as templates,primers were designed and synthesized to amplify rabbit specific transcription factors.Using the brain tissue,trunk tissue and primordial genital crest of 14-16-day-old rabbit fetus as materials,the complete open reading frame sequences of rabbit six specific transcription factors Oct4,Sox2,Klf4,c-Myc,Naong and Lin28 were cloned by RT-PCR.The cloned genes were connected to the eukaryotic expression vector of lentivirus and identified by sequencing.The sequencing results compared with the reference template sequence analysis showed that the matching degree of the cloned specific transcription factors reached 100%,indicating that the specific transcription factors were cloned successfully.The results of sequencing confirmed that the CDS of six specific transcription factors was correctly inserted into the eukaryotic expression vector,and lentivirus Fuw-teto-rOct4,Fuw-teto-rSox2,Fuw-teto-rKlf4,Fu w-teto-rc-My c,Fuw-teto-rNaong and Fuw-teto-rLin28,were successfully constructed to lay the molecular foundation for the next preparation of riPSCs.(3)Study on packaging of lentivirus and infecting rabbit fetal fibroblasts.To build carry rOct4,rSox2,rKlf4,rc-Myc,rNaong,and rLin28 six specific transcription factor carrying hOct4,slow virus vector and buy hSox2,hKlf4,hc-Myc four important specific transcription factor of slow virus carrier for packaging,and separation of in vitro culture of rabbit fetal fibroblasts were infection,induced form riPSCs rabbit fetal fibroblasts reprogramming.Furthermore,the induction efficiency of the two lentiviral vectors and whether the immortalized plasmid pCI-neo-hTERT promoted the formation efficiency of riPSCs were compared.A set of complete induction culture system for riPSCs was screened,and a method to improve the production efficiency of riPSCs was established,which laid a foundation for subsequent riPSCs research.The results showed that lentiviruses packaged by two lentiviral systems from different sources could effectively infect rabbit fetal fibroblasts and form iPSCs clones.The cloning efficiency of six specific transcription factors from rabbit was higher than that of four specific transcription factors from human.However,the addition of pCI-neo-hTERT did not promote the emergence of rabbit iPSCs cloning. |
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