微注射MT-hGH融合基因后小鼠原核卵体外发育和移植的研究

Study on the Development in Vitro and Transfer of Mouse Pronuclear Eggs Following MT-hGH Fusion Gene-Microinjection

将MT-hGH(小鼠金属巯因启动子--人生长激素基因)融合基因,用微注射的方法注入昆明白小鼠原核卵的雄原核中,观察注射外源基因对原核卵的存活,卵裂及2细胞胚胎体外发育的影响。实验结果表明,昆明白小鼠原核卵在雄原核内注射2p1 MT-hGH基因悬液后存活率为86.71％,其存活率与只穿刺雄原核不注射:注射培养液;注射TE缓衡液后存活率相近(分别为84.94％,84.4％和86.42％)。基因注射后原核卵存活率降低主要由注射针机械刺激引起的。原核卵在注射MT-hGH基因后注射胚卵裂率显著降低,以培养24h统计。注射培养液组活胚卵裂率为93.98％,丽注射基因组仅为87.96％(P<0.01)。注射MT-hGH基因组在体外培养条件下,2细胞到胚泡的发育率为40.66％,而注射培养液的对照组为64.09％(P<0.05)。注射基因组胚胎发育速度延缓,部分胚胎延缓12h。将基因注射后的原核卵经体外培养形成的198枚2细胞胚。45枚桑椹胚和161枚胚泡,移植给54只假孕受体,其中16只受体妊娠,产仔49只。目前存活25只。85日龄时。有6只小鼠体重为对照组平均体重的1.207～1.353倍,生长速度较快。经检测其中有5只呈阳性反应为转基因小鼠。

MT-hGH fusion gene were microinjected into male pronueleus of fertilized pronuclear eggs of mice. Gene-microinjected eggs and medium-microinjected control pronuclear eggs were cultured in vitro. Their survival potency, developmental rate and development speed were observed at different times. The results showed that 86.17% of 2Dl MT-hGH gene-microinjected mouse pronuclear eggs surived, 84.49% of medium-microinjeeted con- trol eggs survived. Survival rate of gene-microinjected and medium-microinjected pronuclear eggs have no remarkable difference (P>0.05). After the pronuclear eggs were microinjected with MT-hGH gene the cleavage rate of survival reduced significantly. In the control group and MT-hGH gene-microinjected group the developmental rates of 2-cell to blastocysts were 64.09% and 46.23% respectively. The 2-cell eggs were cultured for 12, 24, 36, 48, 60, 72 and 84 hours. The observational results showed that the developmental rate of gene—microinjected 2-cell eggs was reduced and the developmental speed was delayed. 1982-cell eggs, 45 morulae and 161 blastocysts developed from the gene-microinjected pronuclear eggs were transferred to 45 pseudopregnant recipients. Among them, 16 recipi ents became pregnant and gave birth to 49 offsprings. 25 offsprings lived until now. Among the 25 mice developed from the gene-microinjected embryos, 6 mice were 1.207~1.353 times as heavy as the average body weight of the control mice by 85 days old. They grew relatively rapidly. The integration and expression of MT-hGH gene were analysed in transgenic mice. 5 offsprings showed positive reaction. They were transgenic mice.