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| 1 | Green fluorescent protein (GFP) transgenic pig produced by somatic cell nuclear transfer显示文摘Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm ani-mals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfection system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe-tal-derived fibroblast cells cultured with 4.0 μL/mL liposome and 1.6 μg/mL plasmid DNA for 6 h re-sulted in the highest transfection rate (3.6%). The percentage of GFP reconstructed embryos that de-veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Re-constructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 de-livered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research. | LIU ZhongHua SONG Jun WANG ZhenKun TIAN JiangTian KONG QingRan ZHENG Zhong YIN Zhi GAO Li MA HaiKun SUN Shuang LI YuTian WANG HongBin R S PRATHER | 2008 | Chinese Science Bulletin2008，53，7： | 8 |
| 2 | Generation of cloned calves from different types of somatic cells显示文摘Six types of bovine somatic cell lines, including a granulosa cell line of Chinese red-breed yellow cattle (YGR), a granulosa cell line of Holstein cow (HGR), two skin fibroblast cell lines of two adult Holstein cows respectively (AFB1 and AFB2), a skin fibroblast cell line (FFB) and an oviduct epithelial cell line (FOV) of a Holstein fetus, were established. Somatic cell nu-clear transfer (SCNT) was carried out using these cells as nuclei donor, and a total of 12 healthy calves were cloned. The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated. (i) There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR (33.2% and 35.1%, respec-tively). Pregnancy rates of them were 33.3% and 30.2%, respectively; and birth rates were 16.7% and 11.6%, respectively. (ii) Development rates to the blastocyst stage for SCNT embryos from diffetent individuals (AFB1 and AFB2) differed significantly (27.9% and 39.4%, respectively, P <0.05). Pregnancy rates of them were 36.2% and 36.4%, respectively; and birth rates were 14.9 % and 27.3%, respectively. (iii) There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus (37.9% and 41.5%, respectively, P < 0.05). Pregnancy rates of them were 45.7% and 24.1%, respectively; and birth rates were 22.9 % and 10.3%, respectively. Finally, developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows (HGR, AFB, FFB and FOV) were compared. For in vitro development stage, development rates to the blastocyst stage for SCNT embryos from HGR, AFB, FFB and FOV were 35.1%A, 29.4%B, 37.9%A and 41.5%C, respectively (PABC<0.05); for in vivo development stage, pregnancy rates of them were 30.2%, 36.2%, 45.7% and 24.1%, respectively; and birth rates of them were 11.6%, 17.2%, 22.9% and 10.3% respec-tively. | GONG Guochun1*, DAI Yunping1*, ZHU Huabing2, WANG Haiping1, WANG Lili1, LI Rong1, WAN Rong1, LIU Ying1 & LI Ning1 1. State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, China 2. Institute of Animal Science, Chinese Academy of Agricultural Science, Beijing 100094, China | 2004 | Science China(Life Sciences)2004，47，5： | 5 |
| 3 | 供核细胞对体细胞核移植效率的影响显示文摘利用体细胞核移植技术克隆动物、生产转基因家畜具有极大的应用潜力。然而,核移植效率低下、克隆后代形态异常等问题仍然制约着体细胞核移植技术的产业化进展。影响体细胞核移植效率的因素很多,该文着重从供核细胞的类型、细胞体外培养、细胞凋亡及转基因操作等方面阐述其对体细胞核移植效率的影响。 | 胡艳秋 李文蓉 罗淑萍 | 2006 | 生物技术2006，16，3： | 2 |
| 4 | 转基因和再克隆对克隆胚胎细胞凋亡的影响显示文摘细胞凋亡在着床前胚胎发育过程中发挥着重要作用,胚胎凋亡检测能为获得高质量的体细胞克隆胚胎提供有益信息,进而有助于提高克隆效率.用原位末端标记法对牛的转基因克隆和转基因再克隆囊胚进行了细胞凋亡检测,再克隆囊胚是利用转基因克隆牛的体细胞作为供体细胞进行体细胞核移植后获得的第二代克隆胚胎.结果表明转基因克隆胚胎的囊胚发育率显著低于非转基因克隆胚胎的囊胚发育率,而转基因克隆囊胚的细胞凋亡指数显著高于非转基因克隆胚胎的细胞凋亡指数.此外,转基因再克隆胚胎的囊胚发育率和胚胎细胞凋亡指数与非转基因克隆胚胎的囊胚发育率和胚胎细胞凋亡指数相比差异性不显著.结果表明早期的转基因克隆胚胎发育能力的减弱可能是由于供体细胞的基因转染和药物筛选过程导致的,而再克隆对其早期胚胎的发育能力没有负面影响. | 孙国杰 李荣 戴蕴平 王海平 王莉莉 刘颖 丁方荣 卫恒习 李宁 | 2009 | 自然科学进展2009，19，3： | 1 |
| 5 | Apoptosis of transgenic cloned and recloned bovine blastocysts显示文摘Apoptosis plays an important role in preimplantation embryonic development. Investigating mechanisms of apoptosis can provide useful information for obtaining high-quality embryos and help to improve cloning effciency. Here, we investigated the incidence of blastomere apoptosis in transgenic blastocysts generated by somatic cell nuclear transfer (SCNT) and recloning using a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. Transgenic recloned embryos were the second generation SCNT embryos derived from the somatic cells of a transgenic SCNT calf. The blastocyst rate of transgenic SCNT embryos was lower than that of nontransgenic SCNT embryos. The incidence of apoptosis in transgenic SCNT embryos was higher than that of nontransgenic SCNT embryos. The blastocyst rate and the incidence of apoptosis in transgenic recloned embryos were similar to nontransgenic SCNT embryos. The process of donor cell transfection and drug selection may decrease the developmental capacity of transgenic SCNT embryos. Serial cloning did not influence the developmental capacity of transgenic recloned embryos. | Guojie Sun Rong Li Yunping Dai Haiping Wang Lili Wang Ying Liu Fangrong Ding Hengxi Wei Ning Li | 2009 | Progress in Natural Science:Materials International2009，19，7： | 1 |
| 6 | Clone of Chinese Jinan red-cross yellow cattle and evaluation of reproductive characteristics of cloned calf显示文摘Somatic cell clone technology is a viable ap- proach to preserving endangered livestock and wildlife ge- netic resources. In the present research, somatic cell nuclear transfer (SCNT) was performed using granulose cells from the critical endangered Chinese red-cross yellow cattle as donor cells. A total of 211 oocytes were manipulated and 166 (79%) of them were successfully enucleated. 112 (67.4%) SCNT embryos were reconstructed, 94 (83%) of them cleaved, and 48 (43%) of them developed to blastocyst stage. SCNT blastocysts were transferred to 6 Holstein recipients, and 2 (33%) of them were found to be pregnant. One of them maintained to term and delivered a calf, whereas another aborted. Effect of different fusion buffer (mannitol vs. Zim- merman fusion buffer) and different activation methods (calcium ionophore+6-DMAP vs. cycloheximide+CB) on fu- sion rate and development of SCNT embryos were investi- gated. The results indicated that: (i) on condition of two DC pulses of 2.5 kV/cm for 10 μs each, fusion rates were higher in mannitol solution than in Zimmerman fusion buffer (71% vs. 61%, respectively, p<0.05), but the blastocysts rates did not differ between two treatments (36% vs. 39%, p>0.05 ); (ii) There was no significant difference in development rates to the blastocyst stage for SCNT embryos activated by calcium ionophore+6-DMAP or by cycloheximide+CB (42% vs. 46%, respectively, p>0.05). Microsatellite DNA analysis examining 28 loci confirmed that the cloned calf was genetically identi- cal to the donor Jinan red-cross yellow cattle and different from the recipient females. Growth and reproductive per- formance of cloned cow were evaluated, and there were no difference i cross-red n it between cloned and normal control Jinan yellow cattle. Furthermore, the cloned yellow cow has delivered a healthy yellow calf. | DAI Yunping LIAN Zhengxing ZHU Huabin GONG Guochun WANG Lili WANG Haiping ZHAO Zhihui ZHU Qinghong FEI Jing LI Ning | 2005 | Chinese Science Bulletin2005，50，22： | 0 |
| 7 | 携带人Ⅰ型а1原胶原基因荷斯坦奶牛胚胎的发育研究显示文摘克隆了人Ⅰ型а1原胶原的cDNA基因,将克隆的基因连接到改造后的乳腺特异性表达载体pGC1上,经电穿孔法转染到成纤维细胞中,利用核移植技术获得携带人Ⅰ型а1原胶原基因牛胚胎。最终获得8枚转基因的囊胚,证明用核移植方法获得的携带人Ⅰ型а1原胶原蛋白基因的荷斯坦奶牛胚胎能够正常发育。 | 彭涛 刘婷婷 王亮 | 2009 | 西北农业学报2009，18，2： | 0 |
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