Study of the Efficiency of Chemically Assisted Enucleation Method for Handmade C

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  u0 c1 h# L% rStudy of the Efficiency of Chemically Assisted Enucleation Method for Handmade Cloning in Goat (Capra hircus).
3 M- \5 E) F) M! f0 bAkshey Y, Malakar D, De A, Jena M, Sahu S, Dutta R.8 j. l7 o, X! f
Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
3 j& g' H# s( R' {1.说明：原文来源自（链接）http://onlinelibrary.wiley.com/d ... 1.2010.01732.x/full, O( F5 P3 b+ X; B) o3 F4 b7 I
由干细胞之家新闻小组成员Hyde翻译（转帖请注明）
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0 t/ u4 [: d' n( ]; S9 q* n2.摘要：* F" @& ~& e6 O
我们进行了一项研究，为了发现高效的化学辅助程序用于手工克隆中相关的山羊卵母细胞去核。卵母细胞体外成熟22小时，然后转移到 0.5 μg/ml 的秋水仙胺中孵育2小时。在0.5 mg/ml 透明质酸酶中使用吸管和涡流处理掉卵丘，用链霉蛋白酶处理透明带。8 r$ z: M7 Y" Z5 I: [% ^
极体被拨弄到一侧（Oocytes with extrusion cones were subjected to oriented bisection？），使用微刀（ a micro blade）去除附有极体的三分之一的细胞质。剩余的细胞质作为手工克隆的受体。山羊的成纤维细胞作为细胞核的供体。总体的效率即获得胞质体的量比上使用的卵母细胞的量，明显地（p < 0.05)）高于不使用秋水仙胺的方法(80.02 ± 1.292% vs. 72.9 ± 1.00%, respectively, mean ± SEM). 重组和激活的胚胎在胚胎培养基中培养7天。融合、卵裂、囊胚发育率分别为71.63 ± 1.95%, 92.94 ± 0.91% and 23.78 ± 3.33%，与手工随机去核和非化学辅助（秋水仙胺）去核的效率没有明显的差异性。总结：化学辅助的去核是一项有效和可靠的技术被用于山羊手工克隆，在胞质体选择时摆脱了昂贵仪器的束缚（如倒置荧光显微镜等），避免了可能存在风险的染色体染色和紫外辐射。
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6 `; N# t) B9 n9 J& F" I# }The present investigation was carried out to find an efficient chemically assisted procedure for enucleation of goat oocytes related to handmade cloning (HMC) technique. After 22-h in vitro maturation, oocytes were incubated with 0.5 μg/ml demecolcine for 2 h. Cumulus cells were removed by pipetting and vortexing in 0.5 mg/ml hyaluronidase, and zona pellucida were digested with pronase. Oocytes with extrusion cones were subjected to oriented bisection. One-third of the cytoplasm with the extrusion cone was removed with a micro blade. The remaining cytoplasts were used as recipients in HMC. Goat foetal fibroblasts were used as nuclear donors. The overall efficiency measured as the number of cytoplasts obtained per total number of oocytes used was significantly (p < 0.05) higher in chemically assisted handmade enucleation (CAHE) than oriented handmade enucleation without demecolcine (OHE) (80.02 ± 1.292% vs. 72.9 ± 1.00%, respectively, mean ± SEM). The reconstructed and activated embryos were cultured in embryo development medium (EDM) for 7 days. Fusion, cleavage and blastocyst development rate were 71.63 ± 1.95%, 92.94 ± 0.91% and 23.78 ± 3.33% (mean ± SEM), respectively which did not differ significantly from those achieved with random handmade enucleation and OHE. In conclusion, chemically assisted enucleation is a highly efficient and reliable enucleation method for goat HMC which eliminates the need of expensive equipment (inverted fluorescence microscope) and potentially harmful chromatin staining and ultraviolet (UV) irradiation for cytoplast selection.' S- y$ {8 c& B1 x+ D9 B9 _' j3 X  @; t

0 e0 ^/ a& N4 t&copy; 2010 Blackwell Verlag GmbH.7 m% n% w& [, [* ?/ E

, V6 H) r5 l/ P部分名称和操作技术不太懂，还请各位指点完善。
; B7 l, e: ^7 f8 B2 ^, X个人翻译，理解不准确的语句还望大家积极指出。) ?5 u4 P; n& A3 U0 n8 q: t
下附上原文：

songzxj

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$ Z) v4 h9 `( W不看好手工克隆的发展！第一，这种克隆手段对操作者的技术要求要高很多；第二，效率相对较低，第三，要求较理想的体外培养系统，这对于胚胎体外培养尚不健全的物种，更是难上加难；第四，相对来说显微操作系统的价格已经可以接受，这从国内很多高校研究所闲置的数量可以看出来！！！所以，我认为这种克隆手段可能会局限于经济相对困难的实验室，而不会普及！7 b1 m. w( s) k
一点个人想法而已！