跨胎盘RNAi技术对目的基因下调的研究

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

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7．5d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70ng/μl；注射时间3、5、10、20及30s。]探索获得基因下调胚胎鼠的最适参数，并观察干预48h后胚胎基因型及表现型。结果实验组孕鼠在10μl／g，50ng／μl，5s给药后荧光强度下调最明显，与阴性组、空白组比较干预48h后胚胎存在中枢神经系统、心血管系统异常，实验组胚胎BMPd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。

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 [ intervention solution volume： 5 μl/g, 10 μl/g, 15 μl/g, 20 μl/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 10 μl/g; p/asmid 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 embryos. Conclusion Optimized trans-placental RNAi can be used for the study of genetic function of mice.