跨胎盘RNAi技术对目的基因下调的研究

目的：利用跨胎盘RNA干扰技术下调目的基因，研究小鼠胚胎发育中BMP4基因的功能。方法实验分为Ringer’ s液空白对照组（空白组）、 pSES阴性对照组（阴性组）、 pSES?SiBMP4实验组（实验组），各组孕7．5 d母鼠尾静脉注射干预。分别对干预溶液体积、质粒浓度、注射速度进行研究。依据小鼠克数体重体积分别采用5、10、15及20μl／g；浓度采用10、30、50及70 ng／μl；注射时间3、5、10、20及30 s。]探索获得基因下调胚胎鼠的最适参数，并观察干预48 h后胚胎基因型及表现型。结果实验组孕鼠在10μl／g，50 ng／μl，5 s给药后荧光强度下调最明显，与阴性组、空白组比较干预48 h后胚胎存在中枢神经系统、心血管系统异常，实验组胚胎 BMP4及 Smad4基因 mRNA 下调。结论优化的跨胎盘RNAi技术可用于小鼠基因功能的研究。

Down-Regulation of the Target Genes Using Trans-Placental RNAi

Objective To examine the function of BMP4 in the development of mouse embryo by regulating the target genes with trans?placental RNA interference. Methods The 7. 5?old?day pregnant mice were divided into 3 groups: Ringer ’ s solution group ( blank control group ) , pSES group ( negative group) , and pSES?SiBMP4 group ( experimental group) . Drugs were delivered to the pregnant mice via the tail vein. The volume of intervention solution, plasmid concentration and injection time were analyzed to obtain the optimal parameters for down?regulating the genes inter?vention solution volume: 5μl/g, 10μl/g, 15μl/g, 20μl/g;plasmid concentration: 10 ng/μl, 30 ng/μl, 50 ng/μl, 70 ng/μl;injection time: (3 s, 5 s, 10 s, 20 s, 30 s) . The embryonic genotype and phenotype were detected 48 h after intervention. Results The fluorescence decrease was most obvious in pregnant mice in experiment group when drugs ( intervention solution volume 10μl/g; plasmid concentration 50 ng/μl; injection time 5 s ) were given. Abnormal nervous system and cardiovascular system were found in the experimental mouse embryos 48 hours after intervention, and BMP4 and Smad4 mRNA was found to be down?regulated in experimental mouse embry?os. Conclusion Optimized trans?placental RNAi can be used for the study of genetic function of mice.