基因转录、DNA复制及染色质结构在基因定点修复中的作用及机制研究
摘要
基因治疗是目前治愈人类疾病的最根本方法,但因载体安全性差等问题,其临床应用受到了严重限制。要解决这些问题,首先就是要提高治疗的靶向性,其中最彻底的方法就是原位修复有缺陷的基因,即基因定点修复,它在靶向性、安全性及操作的方便性方面与基因治疗其它方法相比有明显的优势。目前最有希望的定点修复载体是单链寡核苷酸(single stranded oligonucleotide,SSO)。以往的工作证实,SSO确有在多系统多层次上完成基因定点修复的能力。但它的主要缺点是修复效率较低,限制了进一步的应用研究。要想提高其修复效率,当务之急就是揭示SSO介导的基因定点修复的作用机制。
本组已经构建了哺乳动物细胞定点修复的荧光报告系统(F5细胞),并已筛选出了最佳打靶分子(E6)及最佳转染试剂。当把Thymidine作用于F5细胞后,发现修复效率显著提高,因此提出了复制叉渗漏,SSO掺入假说。转录与复制是两个密切联系的过程,尽管转录相关机制研究已取得一定进展,但这些工作都是通过在全基因组范围内调节转录活性而完成的。在这种全局性的调控方式下,最终修复效率的变化是由多种因素改变引起的,不利于进行机制探讨。因此本实验中我们采用了一种特异性的调节靶基因转录的策略,即以靶基因为模板,合成序列特异性的辅助寡核苷酸。因每一条辅助寡核苷酸都可与靶基因上某段特定区域的DNA双链中的一条链互补配对,进而在这个特定的区域内形成一个loop结构。Loop结构形成后,必然会影响到靶基因的转录及复制过程,进而导致修复效率的改变。我们选择了100nt长和25nt长两种长度的辅助寡核苷酸分别与E6一起进行修复实验,发现两组实验的修复效率都有提高。然后,我们把两段25nt长的辅助寡核苷酸作为一个组合,并采用两步转染法进行修复实验,效率提高至对照组的5倍。我们还比较了互补的辅助寡核苷酸的修复效率,结果显示两组的修复效率基本持平。上述实验结果均支持我们设计辅助寡核苷酸的初步设想,即通过形成loop结构,调解靶基因的转录及复制过程,进而改变修复效率。把thymidine和辅助寡核苷酸共同作用于F5细胞后,结果提示最终的修复效率和只用thymidine时的效率基本持平,以这个结果为基础,我们提出了转录复制偶联的修复机制。
丁酸钠作用于F5细胞,修复效率比对照组提高了3倍。当把丁酸钠与辅助寡核苷酸共同作用于F5细胞中,结果显示修复效率比只用辅助寡核苷酸的实验组低。针对此结果,我们认为丁酸钠即可开放染色质结构,又可加快转录速度的特点对定点修复过程既有利又有弊。
本实验中,定点修复过程依旧表现出明显的链偏向性,无论在辅助寡核苷酸或丁酸钠的作用下,总是针对非转录链打靶的分子起效,而与之互补的另一条打靶分子几乎没有修复作用。我们认为此链的偏向性的形成涉及了复制、转录等多种因素的共同作用。
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.
本组已经构建了哺乳动物细胞定点修复的荧光报告系统(F5细胞),并已筛选出了最佳打靶分子(E6)及最佳转染试剂。当把Thymidine作用于F5细胞后,发现修复效率显著提高,因此提出了复制叉渗漏,SSO掺入假说。转录与复制是两个密切联系的过程,尽管转录相关机制研究已取得一定进展,但这些工作都是通过在全基因组范围内调节转录活性而完成的。在这种全局性的调控方式下,最终修复效率的变化是由多种因素改变引起的,不利于进行机制探讨。因此本实验中我们采用了一种特异性的调节靶基因转录的策略,即以靶基因为模板,合成序列特异性的辅助寡核苷酸。因每一条辅助寡核苷酸都可与靶基因上某段特定区域的DNA双链中的一条链互补配对,进而在这个特定的区域内形成一个loop结构。Loop结构形成后,必然会影响到靶基因的转录及复制过程,进而导致修复效率的改变。我们选择了100nt长和25nt长两种长度的辅助寡核苷酸分别与E6一起进行修复实验,发现两组实验的修复效率都有提高。然后,我们把两段25nt长的辅助寡核苷酸作为一个组合,并采用两步转染法进行修复实验,效率提高至对照组的5倍。我们还比较了互补的辅助寡核苷酸的修复效率,结果显示两组的修复效率基本持平。上述实验结果均支持我们设计辅助寡核苷酸的初步设想,即通过形成loop结构,调解靶基因的转录及复制过程,进而改变修复效率。把thymidine和辅助寡核苷酸共同作用于F5细胞后,结果提示最终的修复效率和只用thymidine时的效率基本持平,以这个结果为基础,我们提出了转录复制偶联的修复机制。
丁酸钠作用于F5细胞,修复效率比对照组提高了3倍。当把丁酸钠与辅助寡核苷酸共同作用于F5细胞中,结果显示修复效率比只用辅助寡核苷酸的实验组低。针对此结果,我们认为丁酸钠即可开放染色质结构,又可加快转录速度的特点对定点修复过程既有利又有弊。
本实验中,定点修复过程依旧表现出明显的链偏向性,无论在辅助寡核苷酸或丁酸钠的作用下,总是针对非转录链打靶的分子起效,而与之互补的另一条打靶分子几乎没有修复作用。我们认为此链的偏向性的形成涉及了复制、转录等多种因素的共同作用。
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.
引文
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