Immortalization And Identification Of Ovine Ruminal Fibroblasts Cell

At present,culturing primary ovine ruminal fibroblasts cell(ORFCs)requires a lot of works and complicated procedures.The survival period of cultured ORFCs is short and the number of passages is limited.Therefore,it is necessary to establish immortal ORFCs.This experiment is designed to transfect the PCI-neo-hTERT into ORFCs,so that the foreign hTERT increases to promote the activity of telomerase,thereby maintaining the length of chromosomal telomeres,and increasing the number of divisions and even immortalization of primary cells that die quickly under normal conditions.Using G418 selective medium,positive clones were screened from the transfected ORFCs.The selected positive clones were detected by reverse transcription polymerase chain reaction reverse transcription polymerase chain reaction(RT-PCR)and Western blot(WB)detection methods to detect sheep rumen formation at the gene level and protein level.Transcription and expression of hTERT in fiber cells.Then draw the growth curve of positive clones by hemocytometer counting method.Through the above experiments,we obtained the primary ORFCs,and successfully transferred the plasmid carrying the hTERT gene into the ORFCs to obtain positive cloned cells.Subsequent RT-PCR and WB detected that the hTERT gene expression in the positive cloned cells was significant.By drawing the growth curve,it shows that the immortalized ORFCs have stronger cell division ability.In view of the above experimental results,we believe that this experiment successfully obtained immortalized sheep rumen fibroblasts.It provides an idea for the subsequent use of a single plasmid system to transfect other cells to make them immortal.