大鼠永生化神经前体细胞CDH1小干扰RNA真核载体的构建

Construction of CDH1 small interfering RNA eukaryotic vector in rat immortalized neural progenitor cell and screening of the effective target site

目的构建大鼠永生化神经前体细胞（INPC）CDH1小干扰RNA（siRNA）真核载体。方法根据大鼠CDH1基因的编码序列设计3个小发夹RNA（shRNA）干扰序列及1个阴性对照序列，根据pENTR／H1／TO中间载体说明书设计并合成相应的DNA单链，退火后分别连接到pENTR／H1／TO线性载体上，形成完整载体，提取CDH1 siRNA真核载体后（分别为CDH1 siRNA1、CDH1 siRNA2、CDH1 siRNA3和CDH1 siRNA对照），进行DNA测序鉴定。采用Lipofectamine 2000法，分别将成功构建的CDH1 siRNA真核载体转染大鼠INPC，于转染24h和48h时计算INPC转染效率=INPC成功转染数／细胞总数，于转染48h时提取细胞总RNA，采用实时定量PCR法检测INPC CDH1 mRNA的表达。结果经DNA测序鉴定构建的3个CDH1 siRNA真核载体和1个阴性对照载体所插入的RNA干扰序列均正确，无碱基突变或缺失；INPC转染24h与48h时转染效率分别为（54±5）％和（36±4）％；CDH1 sinNA2转染INPC 48h时INPC CDH1 mRNA表达水平较CDH1 siRNA3降低，且两者均低于CDH1 siRNA1（P〈0．05）。结论本研究成功构建大鼠INPC CDH1 siRNA真核载体。

Objective To construct CDH1 small interfering RNA eukaryotic vector in rat immortalized neural progenitor cell （INPC） and screen the effective target site. Methods Three bands of interfering sequences and one control sequence for CDH1 small hairpin RNA were designed based on CDH1 coding sequence. Following the instructions on pENTR/H1/TO vector, the oligonucleotides were synthesized, annealed and ligated into linearized pENTR/H1/TO vector, respectively. After confirmation by DNA sequencing, positive recombinant plasmids（ CDH1 siRNA1, CDH1 siRNA2, CDH1 siRNA3 and CDH1 siRNAe ）were transfected into INPCs respectively by the liposome method. The pEGFP plasmid was transfected to evaluate the efficiency of transfection. Forty eight hours after transfection, total cellular RNA was extracted and the expression of CDH1 was analyzed by RT-PCR. Results The three eukaryotic vectors for CDH1 siRNA and one control vector were successfully constructed and identified with DNA sequencing. The efficiency of cell transfection was （54±5） % at 24 h after transfection, （36±4） % at 48 h after transfection. The expression of CDH1 mRNA in INPC transfected with CDH1 siRNA2 was lower than that of CDH1 mRNA in INPC transfected with CDH1 siRNA3, and both the expression was lower than that of CDH1 mRNA in INPC transfected with CDH1 siRNA1 at 48 h after transfection. Conclusion The effective small interfering RNA eukaryotic vector for CDH1 in INPC was successfully constructed and screened.