Sparks A E, Canseco R S, Russell C G, Johnson J L, Moll H D, Velander W H, Gwazdauskas F C
Virginia Polytechnic Institute, Blacksburg.
J Dairy Sci. 1994 Mar;77(3):718-24. doi: 10.3168/jds.S0022-0302(94)77005-3.
In vivo fertilized embryos were surgically collected from superovulated dairy cows to evaluate microinjection on embryo development and utilized the polymerase chain reaction technique for selection of transgenic embryos. Seventy-two percent of the embryos with visible pronuclei or nuclei were microinjected with DNA, and the remaining 28% served as uninjected controls. All embryos were cocultured with bovine oviductal epithelial cells. Mean final development scores of embryos within the same initial cell stage at collection were unaffected by microinjection. After 144 h of culture, 45% of the microinjected embryos developed to the morula or blastocyst stage. The transgene was detected in 50, 10, and 9% of demimorulae from embryos microinjected at the 1-, 2-, and 4-cell stages. Frequency of transgene detection was higher in morulae from 1-cell embryos than in morulae from 2- and 4-cell embryos. Use of in vitro coculture, embryo bisection, and polymerase chain reaction technique facilitated selection of bovine embryos that carried the transgene.
通过手术从超数排卵的奶牛体内收集体内受精胚胎，以评估显微注射对胚胎发育的影响，并利用聚合酶链反应技术筛选转基因胚胎。72%具有可见原核或细胞核的胚胎被显微注射了DNA，其余28%作为未注射的对照。所有胚胎均与牛输卵管上皮细胞共培养。收集时处于相同初始细胞阶段的胚胎的平均最终发育评分不受显微注射的影响。培养144小时后，45%的显微注射胚胎发育到桑椹胚或囊胚阶段。在1细胞、2细胞和4细胞阶段显微注射的胚胎的半桑椹胚中，转基因的检测率分别为50%、10%和9%。1细胞胚胎来源的桑椹胚中转基因检测频率高于2细胞和4细胞胚胎来源的桑椹胚。体外共培养、胚胎分割和聚合酶链反应技术的应用有助于筛选携带转基因的牛胚胎。
