Gou Ke Mian, An Xiao Rong, Tian Jian Hui, Chen Yong Fu
The State Key Laboratories for Agrobiotechnology, China Agricultural University, Beijing 100094.
Shi Yan Sheng Wu Xue Bao. 2002 Jun;35(2):103-8.
In this study, the possibility of sheep transgenesis by intracytoplasmic sperm injection (ICSI) was assessed. In experiment 1, activation of ovine oocytes matured in vitro in preparation for ICSI has been investigated with 3.42 mmol/L Ca2+ treatment, ionomycin alone and ionomycin followed by 6-dimethylaminopurine (DMAP) after 3-h delay (group 1, 2 and 3, respectively). After activation, the oocytes were then cultured in SOFaaBSA medium. Cleavage rates were significantly (P<0.05) different among three groups (18.4%, 91.8% and 71.7%, respectively). In additional culture, no parthenotes in group 1, whereas 11% and 17.4% in group 2 and 3 developed to the blastocyst stage. Therefore we used the third activation method in the following ICSI tests. In experiment 2, development of ovine oocytes after ICSI was investigated. Thawed semen from two rams was separated by Percoll centrifugation and was used for ICSI or in vitro fertilization (IVF) trails. A total of 71.8% of oocytes reached the 2-cell stage following living sperm injection, which was significantly (p>0.05) different from those following IVF (41.4%) and sham-ICSI (30.2%). After seven days' culture, no sham-injected oocytes developed into the blastocyst stage, although 7% in ICSI and 16.1% in IVF-oocytes developed into the blastocyst stage, but there was no significant difference in ICSI and IVF groups (p>0.05). In the further study, the possibility of sheep transgenesis by ICSI was assessed. After coinjection of ovine oocytes matured in vitro with dead sperm cold to -20 degrees C and exogenous DNA encoding green fluorescent protein (GFP), seventy-three percent of coinjected oocytes developed to 2-cell stage (33/45) and two of them were transgene-expressing embryos. Among ten embryos at the 16-cell stage, all embryonic cells in one transgenic embryo still expressed GFP. Four coinjected blastocysts were thawed and transferred to the uterine of the two progesterone-synchronized recipient ewe. No pregnancies were detected on the 60th day. These results suggested sheep transgenic embryos could be produced by ICSI and further studies should be performed.
在本研究中，评估了通过胞浆内精子注射（ICSI）进行绵羊转基因的可能性。在实验1中，研究了用3.42 mmol/L Ca2+处理、单独使用离子霉素以及在延迟3小时后用离子霉素加6-二甲基氨基嘌呤（DMAP）处理（分别为第1、2和3组）对体外成熟准备用于ICSI的绵羊卵母细胞的激活情况。激活后，将卵母细胞在SOFaaBSA培养基中培养。三组的卵裂率有显著差异（P<0.05）（分别为18.4%、91.8%和71.7%）。在后续培养中，第1组没有孤雌胚发育，而第2组和第3组分别有11%和17.4%发育到囊胚阶段。因此，在后续的ICSI试验中我们采用了第三种激活方法。在实验2中，研究了ICSI后绵羊卵母细胞的发育情况。从两只公羊采集的解冻精液通过Percoll离心分离，用于ICSI或体外受精（IVF）试验。注射活精子后，共有71.8%的卵母细胞发育到2细胞阶段，这与IVF组（41.4%）和假ICSI组（30.2%）有显著差异（p>0.05）。培养7天后，假注射的卵母细胞没有发育到囊胚阶段，尽管ICSI组有7%、IVF组有16.1%的卵母细胞发育到囊胚阶段，但ICSI组和IVF组之间没有显著差异（p>0.05）。在进一步的研究中，评估了通过ICSI进行绵羊转基因的可能性。将体外成熟的绵羊卵母细胞与冷至-20℃的死精子及编码绿色荧光蛋白（GFP）的外源DNA共注射后，73%的共注射卵母细胞发育到2细胞阶段（33/45），其中两个是转基因表达胚胎。在16细胞阶段的10个胚胎中，一个转基因胚胎的所有胚胎细胞仍表达GFP。将4个共注射的囊胚解冻后移植到两只经孕酮同步化处理的受体母羊子宫内。在第60天时未检测到妊娠。这些结果表明可以通过ICSI产生绵羊转基因胚胎，应进一步开展研究。
