GH/tPA双转基因小鼠的制备及其表达分析

Preparation of GH/tPA double transgenic mice and gene expression analysis

目的旨在获得GH/tPA双转基因小鼠,并比较分析小鼠乳腺中人组织纤溶酶原激活剂(tPA)的表达水平和个体生长发育状况。方法利用2只tPA单转基因雄性小鼠(P03、P05)与经超数排卵处理的正常非转基因雌性小鼠交配,受精卵显微注射线性化的GH质粒、胚胎移植而获得后代小鼠,PCR检测基因整合情况,ELISA和Western blotting检测比较单、双基因表达tPA水平,监测转基因小鼠不同生长阶段的体质量来分析GH基因对小鼠生长发育的影响。结果P03系小鼠共获得286枚受精卵,移植受体母鼠后,顺利产仔77只,经PCR检测获得16只tPA单转基因小鼠(7♂,9♀),13只GH/tPA双转基因小鼠(8♂,5♀),双基因整合率为16.9%;P05系小鼠共获得175枚受精卵,移植受体母鼠后,顺利产仔34只,经PCR检测获得12只tPA单转基因小鼠(5♂,7♀),7只GH/tPA双转基因小鼠(3♂,4♀),双基因整合率为20.6%。单、双转基因小鼠乳腺中表达tPA的最高量分别为82.5μg/mL、674μg/mL,GH/tPA双转基因小鼠乳腺中表达tPA的量明显高于tPA单转基因(P<0.05)。在小鼠的整个生长周期内,单、双转基因与正常非转基因小鼠的体质量增长均没有显著的差异(P>0.05)。结论本实验成功地制备了GH/tPA双转基因小鼠,结果表明GH基因导入小鼠体内能够显著地提高目的基因tPA的表达水平,且不会对转基因小鼠的生长发育造成任何明显的影响,这为将来制备高表达转基因动物生产医药蛋白及其育种奠定了基础。

Objective To obtain GH/tPA double transgenic mice,analyze the expression level of tissue plasminogen activator(tPA)in the mammary glands and observe the growth and development of the transgenic mice.Methods We obtained the offspring mice of 2 tPA single transgenic mice(P03 and P05)mated with a female nontransgenic mouse by microinjection of linearized GH plasmid into the fertilized eggs and embryo transfer.PCR was used to detect the gene integration.The expression levels of tPA in single gene and double gene transgenic mice were compared using ELISA and Western blotting.We assessed the effects of GH gene transduction on the growth and development of the transgenic mice by observing body weight changes of the mice at each developmental stage.Results A total of 286 fertilized eggs were collected from P03 mice,and after embryo transfer,77 offspring mice were obtained,including 16 tPA single transgenic mice(7 male,9 female)and 13 GH/tPA double transgenic mice(8 male,5 female)as confirmed by PCR.The integration rate of the double genes was 16.9%.A total of 175 fertilized eggs were collected from P05 mice,and 34 offspring mice were obtained including 12 tPA single transgenic mice(5 male,7 female)and 7 GH/tPA double transgenic mice(3 male,4 female),in which the integration rate of the double genes was 20.6%.The highest expression level of tPA in the mammary gland was significantly higher in double than in single transgenic mice(674μg/mL vs 82.5μg/mL,P<0.05).In the whole growth cycle of the mice,no significant difference in weight gain was observed in the single or double transgenic mice as compared with the naïve mice(P>0.05).Conclusion We successfully prepared GH/tPA double transgenic mice,in which GH gene transduction significantly increases the expression level of target gene tPA without affecting the growth and development of the transgenic mice.This success suggests a promising approach to preparing transgenic animals for producing pharmaceutical proteins and the breeding of the transgenic animals.