用于筛选顺式反应元件的慢病毒启动子报告载体随机文库的构建

Construction of lentivirus based promoter reporter random library for screening context dependent cis-elements

目的建立含有随机序列与miniCMV融合的启动子,驱动抗生素耐药基因的表达;用于筛选和寻找不同处理条件下的反应性元件。方法设计针对Puromycin抗性基因,即嘌呤霉素N-乙酰转移酶基因(puromycin N-acetyltransferase gene,PAC)的引物,扩增目的条带并通过PacI+Pme1限制性内切酶克隆至目的载体pWPI;然后,设计并合成接头序列+20N随机序列+miniCMV的融合片段,通过PCR的方法得到双链序列,并进行酶切,通过Cla1+PacI克隆入前述pWPI-PAC载体。克隆得到的载体组合即为慢病毒反应元件筛选报告载体文库。在此基础上,我们将报告载体文库转染细胞,并进行超声照射。超声照射剂量为10min,100mW/cm2。每天一次,一共照射3天。每次超声照射后,对细胞进行puromycin处理。4天后,收集超声照射存活细胞,提取DNA,PCR扩增含有20N的序列并进行测序鉴定。结果我们成功设计了一套文库构建方案,初步构建了20N随机序列+miniCMV+Puromycin抗性基因的文库。将该文库转染细胞,超声照射后可以增加转染有对超声反应克隆细胞中抗性基因表达;我们通过获取Puromycin处理后的存活细胞,发现了潜在的对超声反应的调控元件。结论我们构建的20N随机序列+miniCMV+Puromycin抗性基因文库,可以用于筛选对超声等特定条件有表达调控反应的顺式反应元件;进而为该元件控制基因表达提供依据。

Objective To construct a lentivirus based library containing a fused promoter of 20N random sequence plus miniCMV, which drives the puromycin resistant gene PAC expression, for screening of response elements under specific contexts. Methods Primers specific for puromycin resistant gene PAC was designed and the amplicons were a- chieved via PCR. Then the amplieons were cloned into the lentivirus vector pWPI with the help of restriction enzyme Pac I and Pme I. An adaptor sequence plus 20N random sequence together with miniCMV sequence were designed and synthe- sized. The complementary strand of the synthesized sequence was achieved by PCR reaction with the help of a primer. The obtained double strand DNA were then cloned into the above pWPI-PAC vector, generating the library. The library was transfected into Hela cells and the cells were subjected to ultrasound irradiation, with the parameter of 100mW/cm2 for 10 minutes each day and continued for 3 days. The processed cells were additionally treated with puromycin and the DNA in the survived cells at day 4 were isolated and the exact sequence of the 20N in these cells were identified by se- quencing. Results We here proposed a strategy to construct random promoters driving selection gene expression. The constructed reporter library contained 20N, miniCMV and PAC gene. Under ultrasound irradiation, these cloned contai- ning promoters upregulated by ultrasound drives the PAC gene expression and thus more resistant to puromycin induced cell death. The detailed information of these promoters were able to be characterized by sequencing the 20N in the sur- vived ceils by sequencing the amplieons corresponding to the 20 N. Conclusion We have successfully constructed a lenti- virus based random library, in which puromycin resistant gene PAC was driven by random fused promoters. Together with puromyein treatment, the library can be applied for identification of specific cis-elements under certain contexts. What＇s more, these identified cis-elements could be promising in controlling spatiotemporal gene expression.