Abstract:

【Objective】The integration of the double genes is able to promote the expression level of the target genes in the transgenic organisms. The aim of this study was to obtain the rhPA/GH double transgenic rabbits, and then the expression level of the target gene rhPA and the individual growth and development of these rabbits were compared and analyzed, so as to provide a new new approach for preparation of high expression rhPA transgenic animals and genetic breeding. 【Method】 Not Ⅰ/ Sal Ⅰ double enzyme digestion PCL25/GH plasmid and QIAGEN DNA gel purification kit were used to recover gene fragments for microinjection. The three rhPA single-transgenic rabbits (K06, K10 and K17) were used as donors. The rhPA/GH double-transgenic rabbits were obtained by FSH/hCG superovulation, pronuclear microinjection of fertilized eggs, embryo transfer, phenol / chloroform extraction of newborn rabbit’ ear tip tissue genome and PCR integrated detection. In addition, the expression levels of rhPA in single-transgenic rabbit and double-transgenic rabbit whey were compared by ELISA and Western blotting. The body weight of transgenic rabbits at different months was measured, and the effects of GH on growth and development of rabbits were analyzed by body weight at growth stage. 【Result】The about 16 700 bp of microinjection gene fragments were successfully obtained. A total of 122 eggs were obtained from 3 donor rabbits, 103 of which were fertilized, and the fertilization rate was 84.4% (103/122). After microinjection, 81 fertilized eggs with good morphology were selected and transplanted into 6 recipient rabbits by synchronous estrus. Five rabbits were pregnant and the pregnancy rate was 83.3% (5/6). A total of 32 offspring were born at the end of pregnancy. There were 19 rhPA transgenic rabbits identified by PCR, and 11 of them were the rhPA/GH double transgenic rabbits (7♂, 4♀), so the double-gene integration rate was 34.4% (11/32). The four rhPA/GH double-transgenic female rabbits were derived from K06 donor rabbits (labelled K06-1 and K06-2), K10 donor rabbit (labeled K10-1) and K17 donor rabbit (labeled K17-1), respectively. The rhPA expression in single transgenic rabbit whey of No. K06 was 42.2 μg·mL ^-1, and the rhPA expression in the double transgenic rabbit whey of No. K06-1 and K06-2 was 432 and 444 μg·mL ^-1, respectively. The rhPA expression in the single transgenic rabbit whey of No. K10 was 42.8μg·mL^-1, and the rhPA expression in the double transgenic rabbit whey of No. K10-1 was 636 μg·mL ^-1. The rhPA expression in single transgenic rabbit whey of No. K17 was 15.2μg·mL^-1, and which in double transgenic rabbit whey of No. K17-1 was 248 μg·mL ^-1. The expression of rhPA in mammary glands of four female rhPA/GH double-transgenic rabbits (K06-1, K06-2, K10-1, and K17-1) were 248-636 μg·mL^-1, which was much higher than that of rhPA single-transgenic rabbits (K06, K10, K17, expression levels: 15.2-42.8μg·mL ^-1). That is to say, the expression level significantly increased about 10.2 to 16.3 times, and it showed that GH could synergistically promote the expression level of target gene rhPA in the mammary glands of transgenic rabbits. Besides, the western blotting results showed a band of about 39.0 kDa, which was the same size as the target protein rhPA, further proving that this protein expressed in the breast of transgenic rabbits was the target product rhPA. By measuring the weight of rhPA/GH double-transgenic rabbits from their birth to 6 months continuously, it was found that the weight was no significant difference between the rhPA/GH transgenic rabbits and the normal non-transgenic rabbits. The growth curve was drawn to further indicate that 4 transgenic rabbits with integrated GH and 2 normal rabbits without integrated GH had no significant difference in body weight at different stages of growth and development. The body weight for 6 months was about 4.0-5.0 kg. This proved that the introduction of GH did not have harmful influence on their survival and normal growth and development to adulthood. 【Conclusion】In this experiment, rhPA/GH double-transgenic rabbits were successfully prepared, and it has been proved that the introduction of GH could significantly increase the expression level of rhPA. Moreover, it could not affect the growth and development of transgenic rabbits. This laid a foundation for the preparation of high expression transgenic rabbits and other animals in the future, and also provided a new idea and method for the establishment of transgenic animal mammary gland bioreactors and transgenic breeding..

Key words: double transgenic rabbits, expression, superovulation, microinjection, growth curve