Abstract: 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). 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.

Key words: double transgenic rabbits; expression; superovulation; microinjection; gene integration