Hirabayashi M, Hirao M, Takahashi R, Kimura K, Hirasawa K, Ueda M, Hochi S
YS New Technology Institute Inc, Shimotsuga-Gun, Tochigi, Japan.
J Vet Med Sci. 2000 Oct;62(10):1047-52. doi: 10.1292/jvms.62.1047.
Superovulation of female rabbits was induced by subcutaneous injection(s) of porcine FSH. Zygotes were recovered 17 to 19 hr after hCG injection and were classified into two categories under a microscope equipped with Nomarski interference-contrast optics at x 200 magnification: (A) zygotes with clearly visible pronuclei, or (B) zygotes with visualized pronuclei after 10 min centrifugation at 12,000 x g. No significant difference between strains was found in the proportion of category-A zygotes (JW 72.6% vs NZW 79.3%). Pronuclei of category-A zygotes were located in the center of the cytoplasm, and the pronuclei of category-B zygotes were slightly moved by centrifugation toward the mass of cytoplasmic lipid droplets. Exogenous DNA solution (5 microg/ml of fusion gene composed of bovine alphaS1-casein promoter and human growth hormone structural gene) was microinjected into the pronucleus of the JW zygotes. The pronucleus of category-A zygotes with a mean volume of 7.4 pl swelled up to 16.6 pl (132% increase), while that of category-B zygotes with a mean volume of 6.1 pl swelled up to 15.9 pl (148% increase). Nevertheless, similar proportions of category-A and category-B zygotes developed into offspring after transfer to recipient females (11.1 and 11.2%, respectively). The efficiency to produce hGH-carrying transgenic rabbits was 0.9% (2/235) from category-A zygotes and 0.5% (1/215) from category-B zygotes (P>0.05). To date, transgenic rabbits have been produced without centrifugation of pronuclear zygotes. However approximately 25% of fertilized rabbit zygotes can be used for DNA microinjection after they have been centrifuged to visualize their pronuclei.
通过皮下注射猪促卵泡素诱导雌性兔超排卵。在注射人绒毛膜促性腺激素（hCG）后17至19小时回收受精卵，并在配备诺马斯基干涉相差光学系统的显微镜下以200倍放大率将其分为两类：（A）具有清晰可见原核的受精卵，或（B）在12,000×g下离心10分钟后可见原核的受精卵。在A类受精卵的比例上，品系间未发现显著差异（JW为72.6%，NZW为79.3%）。A类受精卵的原核位于细胞质中心，B类受精卵的原核经离心后向细胞质脂滴团块稍有移动。将外源DNA溶液（由牛αS1-酪蛋白启动子和人生长激素结构基因组成的融合基因，浓度为5微克/毫升）显微注射到JW品系受精卵的原核中。平均体积为7.4皮升的A类受精卵原核膨胀至16.6皮升（增加132%），而平均体积为6.1皮升的B类受精卵原核膨胀至15.9皮升（增加148%）。然而，将A类和B类受精卵移植到受体雌性兔后，发育成后代的比例相似（分别为11.1%和11.2%）。从A类受精卵产生携带人生长激素（hGH）转基因兔的效率为0.9%（2/235），从B类受精卵产生的效率为0.5%（1/215）（P>0.05）。迄今为止，未对原核受精卵进行离心就已培育出转基因兔。然而，约25%的受精兔受精卵在离心以使其原核可见后可用于DNA显微注射。
