Krisher R L, Gibbons J R, Canseco R S, Johnson J L, Russell C G, Notter D R, Velander W H, Gwazdauskas F C
Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061-0315.
Transgenic Res. 1994 Jul;3(4):226-31. doi: 10.1007/BF02336775.
The effect of DNA microinjection at various times after in vitro insemination on DNA detection and survival rates of bovine embryos was investigated. Oocytes were inseminated 24 h after maturation with frozen/thawed semen prepared with a Percoll separation procedure. At 11, 15 and 19 h after insemination, embryos were centrifuged to visualize pronuclei and microinjected with a murine whey acidic protein-human protein C genomic DNA construct. After culture for 7 days on Buffalo Rat Liver cells, embryos were assessed for stage of development and assayed for the presence of the transgene by polymerase chain reaction. Of zygotes in the 11 h after insemination treatment, 16% (25/152) of non-injected and 7% (11/161) of injected embryos developed to the morula or blastocyst stage. Comparable development of non-injected and injected embryos treated at 15 h after insemination was 15% (23/158) and 4% (6/159) and treated at 19 h after insemination was 14% (23/162) and 1% (1/165), respectively. Development of injected embryos was greater (p < 0.05) when injection was performed at 11 h after insemination compared to 19 h after insemination. Development of non-injected embryos was greater (p < 0.01) than that of injected embryos. There was no difference in transgene detection frequency in embryos of all developmental states between treatments (53% at 11; 50% at 15; 48% at 19 h after insemination). Injected embryos testing positive for the presence of the transgene exhibited increased development over negative embryos (p < 0.01). Greater development efficiencies can be obtained in microinjected bovine embryos when injection is performed early in pronuclear formation.
研究了体外受精后不同时间进行DNA显微注射对牛胚胎DNA检测及存活率的影响。成熟24小时后的卵母细胞用经Percoll分离程序制备的冻融精液进行受精。受精后11、15和19小时，将胚胎离心以观察原核，并显微注射鼠乳清酸性蛋白-人蛋白C基因组DNA构建体。在水牛大鼠肝细胞上培养7天后，评估胚胎的发育阶段，并通过聚合酶链反应检测转基因的存在。在受精后第11小时处理的受精卵中，未注射的胚胎有16%（25/152）发育到桑椹胚或囊胚阶段，注射的胚胎有7%（11/161）发育到该阶段。受精后第15小时处理的未注射和注射胚胎的可比发育率分别为15%（23/158）和4%（6/159），受精后第19小时处理的分别为14%（
