Chauhan M S, Nadir S, Bailey T L, Pryor A W, Butler S P, Notter D R, Velander W H, Gwazdauskas F C
Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061-0315, USA.
J Dairy Sci. 1999 May;82(5):918-26. doi: 10.3168/jds.S0022-0302(99)75310-5.
The present study was carried out to 1) evaluate the viability of in vitro fertilized zygotes after microinjection of DNA, 2) assess the influence of oocyte quality upon the development rate of embryos when injected with DNA, and 3) determine the integration frequency of green fluorescent protein DNA into microinjected embryos. Oocytes were aspirated from ovaries of nine nonlactating Holsteins and were categorized into grades A, B, C, and D. At 16 h after in vitro fertilization, approximately half of the pronuclear stage presumptive zygotes were classified as having 1 pronucleus or 2 pronuclei, and they were microinjected with DNA constructs. A potential predictor of DNA integration frequency at d 10 was assessment of the incidence of green fluorescing embryos. The proportion of cleaved embryos that developed to morulae or blastocysts was not different between groups with 1 pronucleus injected (45%), 1 pronucleus uninjected (64%), or 2 pronuclei injected (49%). However, the development of morulae or blastocysts was higher in the group with 2 pronuclei uninjected (69%). The overall developmental score of green fluorescent protein-positive embryos was higher for grade A oocytes (1.3 +/- 0.1) than for grade B (0.8 +/- 0.1), C (0.6 +/- 0.1), or D (0.3 +/- 0.1) oocytes. The results show that production of transgenic bovine blastocysts can occur from the microinjection of a presumptive zygote having only one visible pronucleus. Initial oocyte quality is an important factor in selection of oocytes suitable for microinjection of DNA and for preimplantation development to produce bovine transgenic embryos.
1）评估显微注射DNA后体外受精合子的活力；2）评估注射DNA时卵母细胞质量对胚胎发育率的影响；3）确定绿色荧光蛋白DNA整合到显微注射胚胎中的频率。从9头非泌乳荷斯坦奶牛的卵巢中抽吸卵母细胞，并将其分为A、B、C和D级。体外受精16小时后，大约一半的原核期假定合子被分类为具有1个原核或2个原核，然后对它们进行DNA构建体的显微注射。在第10天，绿色荧光胚胎的发生率评估是DNA整合频率的一个潜在预测指标。注射1个原核的组（45%）、未注射1个原核的组（64%）或注射2个原核的组（49%）之间，发育至桑椹胚或囊胚的分裂胚胎比例没有差异。然而，未注射2个原核的组中桑椹胚或囊胚的发育率更高（69%）。A级卵母细胞来源的绿色荧光蛋白阳性胚胎的总体发育评分（1.3±0.1）高于B级（0.8±0.1）、C级（0.6±0.1）或D级（0.3±0.1）卵母细胞来源的胚胎。结果表明，仅具有一个可见原核的假定合子的显微注射可产生转基因牛囊胚。初始卵母细胞质量是选择适合DNA显微注射和用于产生牛转基因胚胎的植入前发育的卵母细胞的重要因素。
