转K2.9基因绒山羊体细胞核移植技术体系的优化研究

doi:10.3864/j.issn.0578-1752.2012.10.020

Abstract

Abstract: 【Objective】In order to change the protein composition of wool and improve the quality of wool, transgenic somatic cell nuclear transfer technology was used to prepare K2.9 (gene from hair keratin intermediate filament type Ⅱ) transgenic cashmere goat embryos to produce transgenic goats. 【Method】 Cashmere goat fetal fibroblast cells were transfected by hair follice-specific expression vector pcDNA3.1-K containing K2.9 and Neor gene, and G418 was used to select transgenic cells as donor cells. To improve the efficiency of nuclear transfer technology, the effects of the activation methods and the sources of donor cells and oocytes on the development of parthenogenetic embryos were studied. Identification of the genomic DNA of transgenic blastocysts by ploymerase chain reaction (PCR) has proved that the exogenous gene had already been integrated into genomes of blastocyst cells. 【Result】 The cleavage rate of adult cashmere goat parthenogenetic embryos was significantly increased by Iono+6-D activation. Iono+6-D was more suitable to the activation of adult cashmere goat oocytes than A23187+6-D. The cleavage rate of lamb pathenogenetic embryos was significantly lower than that of adult cashmere goat parthenogenetic embryos, but the blastocyst rate had no significant difference. The development of nuclear transfer embryos from the transgenic fibroblast cells of two cashmere goat fetuses as donor cells weren’t affected, but the fusion rate of nuclear transfer embryos from the transgenic fibroblast cells of 2nd cashmere goat fetuse as donor cells were greatly increased. The development of nuclear transfer embryos from lamb oocytes was signicicantly reduced. The manipulated blastocysts were confirmed carrying K2.9 gene by PCR. 【Conclusion】The transgenic somatic cell nuclear transfer technology could produce cashmere goat blastocysts carrying K2.9 gene , which were constructed through K2.9 transgenic cashmere goat fetal fibroblast cells by hair follicle-specific expression vector pcDNA3.1-K as donor cells and adult cashmere goat oocytes as receptors, and activated by Iono+6-D.

Key words: Cashmere goat, hair keratin intermediate filament type Ⅱ, fetal fibroblast cells, transgene, somatic cell nuclear transfer

PAN Xiao-Yan, YU Yong-Sheng, LIU Xiao-Hui, WANG Zheng-Chao, WANG Xiao-Yang, PU Qing-Lin, ZHANG Li-Chun, JIN Hai-Guo. Construction of Cashmere Goat Embryos Carrying K2.9 Gene by Transgenic Somatic Cell Nuclear Transfer Technology[J].Scientia Agricultura Sinica, 2012, 45(10): 2067-2075.

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Construction of Cashmere Goat Embryos Carrying K2.9 Gene by Transgenic Somatic Cell Nuclear Transfer Technology

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