Study On Introduction And Integration Of α-1, 3-GT Silencing Gene Microjected To Mice

Through putting human H1 promoter before the shRNA reported,a siRNA sequence was designed to silenceα-1,3-GT gene in murine. Subsequently a series of primer segments were devised and joined by PCR.After the connected product was cloned into the pGEM-T vector, a vector that can silence the murineα-1,3-GT gene was acquired.After linearization by enzym e digestion,the vector silencingα-1,3-GT gene was transferred into murine fertilized eggs and carry out embryo transplantation.Finally,the integration of exogenous gene was examined by PCR and Southern blot analysis after the birth of young mice.This reasearch will help us to constitute an important foundation for acquiringα-1,3-GT knock down mice and mouse model for xenotransplantation research.1.Fragments of devised primer have been joined successfully by two-step PCR and cloned into pGEM-T vector by PCR and sequencing.2.Linear vector for silencingα-1,3-GT gene was microjected into 2846 of murine ferti- lized eggs and 2291 of useful embryos were transferred into 76 of recipients.35 of them had pregnancies.The pregnant is 46.1%.There were 29 of mother mice aborting and the others giving birth to 21 offsprings in the gestation period.3.The influence that the number of embryo and different cell stages acted on lambing rate was observed in the process of microjection.The results showed:the group of 21-30 embryos have significantly higher dropping rate than other groups;Two-cell stage has signifi- cantly higher conception rate and dropping rate than one-cell stage.4.The integration of exogenous gene was examined in the 21 of live mice and 17 of dead mice(including fetal death and mummy)by PCR and Southern blot analysis. Seven of positive mice were observed including three of live mice and four of dead mice.The total integrated rate of exogenous gene is 14.3%...