Krisher R L, Gibbons J R, Gwazdauskas F C
Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061-0315, USA.
J Dairy Sci. 1995 Jun;78(6):1282-8. doi: 10.3168/jds.S0022-0302(95)76748-0.
Bovine embryos that had been microinjected with DNA were examined for their potential use as donor embryos in nuclear transfer. Donor embryos were obtained from oocytes collected by transvaginal oocyte aspiration, matured and fertilized in vitro, microinjected with a murine whey acidic protein-human protein C genomic DNA construct, and cultured in vitro on liver cells of buffalo rat (Rattus norvegicus). Blastomeres from these embryos were transferred into enucleated bovine oocytes received from an abattoir by electrofusion at 40 h postmaturation. Following 7 d of culture, the developmental stage was recorded, and resulting embryos were prepared for analysis by polymerase chain reaction. Embryos that were derived from microinjected donor embryos did not differ from control donor embryos (11 vs. 8.6%) in development to the morula and blastocyst stage. Of the biopsies from 20 microinjected donor embryos, 19 were positive for the injected DNA. Of 37 embryos developing normally, only 12 (32.4%) were positive for the injected DNA. These results indicate that microinjected embryos can be successfully used in a nuclear transfer program to produce additional viable embryos and that these embryos may be reliably screened for the transgene for transfer to recipients.
对已显微注射DNA的牛胚胎进行检查，以评估其作为核移植供体胚胎的潜在用途。供体胚胎从经阴道卵母细胞抽吸收集的卵母细胞中获得，在体外成熟并受精，用鼠乳清酸性蛋白-人蛋白C基因组DNA构建体进行显微注射，然后在水牛大鼠（褐家鼠）的肝细胞上进行体外培养。在成熟后40小时，通过电融合将这些胚胎的卵裂球转移到从屠宰场获得的去核牛卵母细胞中。培养7天后，记录发育阶段，并对所得胚胎进行聚合酶链反应分析准备。源自显微注射供体胚胎的胚胎在发育到桑葚胚和囊胚阶段方面与对照供体胚胎没有差异（分别为11%和8.6%）。在对20个显微注射供体胚胎进行的活检中，19个对注射的DNA呈阳性。在37个正常发育的胚胎中，只有12个（32.4%）对注射的DNA呈阳性。这些结果表明，显微注射的胚胎可以成功用于核移植程序以产生更多有活力的胚胎，并且这些胚胎可以可靠地筛选转基因以便移植给受体。
