目的:获得GH/tPA双转基因小鼠,分析组织型纤溶酶原激活剂(tPA)在乳腺中的表达水平,观察转基因小鼠的生长发育。
方法:我们通过将线性化GH质粒显微注射入受精卵并进行胚胎移植,获得了2tPA单转基因小鼠(P03和P05)与雌性非转基因小鼠交配的后代小鼠。用PCR检测基因整合。采用ELISA和Westernblotting方法比较tPA在单基因和双基因转基因小鼠中的表达水平。我们通过观察小鼠在每个发育阶段的体重变化来评估GH基因转导对转基因小鼠生长发育的影响。
结果:总共从P03小鼠中收集了286个受精卵,胚胎移植后,获得77只后代小鼠,包括16只tPA单转基因小鼠(7只雄性,9只雌性)和13只GH/tPA双转基因小鼠(8只雄性,5雌性),如通过PCR确认。双基因的整合率为16.9%。总共从P05小鼠中收集了175个受精卵,并获得了34只后代小鼠,其中包括12只tPA单转基因小鼠(5只雄性,7只雌性)和7只GH/tPA双转基因小鼠(3只雄性,4女),其中双基因的整合率为20.6%。tPA在乳腺中的最高表达水平是单个转基因小鼠的两倍(674μg/mL比82.5μg/mL,P<0.05)。在小鼠的整个生长周期中,与正常小鼠相比,单转基因或双转基因小鼠的体重增加没有显着差异(P>0.05)。
结论:我们成功制备了GH/tPA双转基因小鼠,其中GH基因转导显着增加了靶基因tPA的表达水平,而不影响转基因小鼠的生长发育。这一成功表明了制备用于生产药物蛋白质的转基因动物和转基因动物的育种的有希望的方法。
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 (674 μg/mL vs 82.5 μg/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 na?ve mice (P>0.05).
CONCLUSIONS: 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.