Gene Transcription, Dna Replication And Chromatin Structure In Gene Designated Repair And Mechanism Study

Gene therapy is the most thorough method for the treatment of human diseases,but its application has been seriously limited for the insecurity of gene transfer vectors.To solve the problem,the most simple way is to correct the mutated gene precisely,that is targeted gene repair.Compared with other methods,targeted gene repair has obvious advantages in precision,safety and simplicity.And the most promising vehicle in this field is the single-stranded DNA oligonucleotide(SSO).Although SSO can complete the gene correction in multi-levels and multi-systems,its efficiency is too low to perform further application.It is important to clarify the repair mechanism to improve the correction efficiency.The fluorescent reporter system in mammalian cells has been established in our lab,including HeLa-mEGFP cell clone(F5 cell),targeting molecular (E6),as well as the suitable transfection reagent.When thymidine was added into F5 cells,the repair efficiency was improved obviously,so we proposed the hypothesis of replication fork leakage,SSO incorporation. Transcription and replication are two close related processes,although the mechanism on transcription level has been elucidated in certain degree, but it has all been done by changing transcription activity in the whole genome.So the change of repair efficiency may be induced by multi-variable factors.In this research,we designed the experiments by using assistant oligonucleotides to regulate the transcription of target gene.Because each assistant oligonucleotide can incorporate into the DNA double strands and pair with its complementary sequence,and then a loop structure is formed in this region.The loop structure can affect the transcription and replication of target gene to improve the repair efficiency.We selected the assistant oligonucleotides of 100nt and 25nt with one step transfection,and found that both of them could improve the repair efficiency up to three times.When we combined two assistant oligonucleotides of 25nt in one group,and adopted the two steps transfection method,the efficiency was five times higher than the control with E6 alone.No obvious difference of efficiency was observed when the two complementary assistant oligonucleotides were used separately to F5 cells.The above results suggested that the loop structure induced by assistant oligonucleotides may regulate the replicaion,transcription and repair process of targeted gene to improve the efficiency.The repair efficiency was almost the same as using thymidine alone when the assistant oligonucleotides were used together to transfer F5 cells,then we proposed the replication transcription coupled mechanism.When sodium butyrate was incubated with F5 cells,the repair efficiency was three times higher than the control with E6 alone.But when it combined with the assistant oligonucleotides to the same cell line, the repair efficiency was even lower than that with assistant oligonucleotides alone,suggesting that sodium butyrate has two kinds of effect on repair reaction.It can relax chromatin structure to facilitate the repair process and make transcription fast to decrease the repair efficiency.The strand bias was also observed in our experiment,which means that the sense SSO is always more effective than the anti-sense SSO.And we believed that this strand bias is related to multi-factors affecting transcription and replication process.