山羊外源基因转移技术

TECHNOLOGY OF FOREIGN GENE TRANSFER INTO GOAT

对24头供体山羊进行超数排卵处理,16头超排有效。在第1次配种后48h左右取卵,共收集到卵155个。回收率79.08％(155/16)。平均每头供体排卵12.25个(196/16)。在Normarski干涉相差显微镜下观察65个单细胞受精卵及29个2～4细胞期胚胎的原核及细胞核,并分别注射人生长激素基因(p^(SMGHI)/BamHI),其中91枚移植到经同期发情处理的14头受体中。结果7头妊娠,共产活羔11头,死胎1头。经用Digoxigcnin标记核酸探针对转移基因羊检测,4头整合了外源基因。整合频率为33.33％(4/12)。采用酶联免疫吸附法(ELISA)对仍存活的2头未整合、2头整合羊检测是否表达外源基因,全为阴性。

24 female Goats were superovulated with Gonadotrophins. About 48h after first mating,total 155 ova were recovered from 16 stimulated donors. The ova recovery rate was 79.08％(155/196).The average number of ova produced by per donor was about 12.25％(196/16). Pronuclei or nuclei in atotal of 65 one-celled zygotes and 29 two-four cell stage embryos were visualized under Normarski IC microscope, and microinjected with Human Growth Hormone Gene(p^(SMGHI)/BamHI) respectively. 91 injected zygotes and embryos were transferred surgically into 14 synchronized recipients. After embryotransfer, 7 recipients became pregnant, and total 12 lambs were born, 11 alive and one dead at parturition.The frequency of hGH integration was examinated by using DNA probes labeled with NonradioactiveDigoxigenin. Results showed that 4 in 12 tested lambs carried the foreign genes, the integration frequencywas 33.33％(4/12). Gene expression in 4 living lambs (2 hGH gene integration positive, 2 negative) wasdetected by ELISA, and no one exprcsscd humane Growth Hormone in plasma.