摘要
目的 在小鼠胚胎干细胞上进行α 1,3半乳糖基转移酶(α GT)基因打靶。 方法 利用长距离PCR 和套式PCR从C57小鼠基因组DNA中克隆出α GT基因组DNA片段,构建了针对α GT基因外显子4的置换型基因 打靶载体ploxp GT,同源长臂(5.0kb),同源短臂(3.97kb),同源序列全长共约9.0kb。将打靶载体酶切线性化并以 电穿孔方法转移入小鼠MEES细胞。 结果 通过G418和Gancyclovir正负选择出171株ES细胞药物抗性克隆, Southernblot鉴定获得11个α Gal(+ -)小鼠ES细胞克隆,重组频率为6.4%。 结论 上述方法能够完成α GT 基因敲除,为进一步完成α GT基因敲除小鼠奠定了基础。
Objective To make α-GT gene targeting in mouse embryonic stem cell and study the role of α-Gal on xenotransplantation. Methods The long arm(5.0?kb) and short arm(3.9?kb) in α-GT gene were isolated from the genomic DNA of C57B/6J mouse by long polymerase chain reaction(PCR) and nested PCR.These fragments were cloned into ploxP vector on both sides of the neomycin gene.Then,we obtained a gene targeting vector ploxP-GT.The Not Ⅰ-linearized targeting vector was electroporated into C57BL/6J mouse ES cells. Results After selection for G418 and gancyclovir resistance,11 of 171(6.4%) resistant clonies were identified by Southern blot analysis to contain a 6.4?kb EcoRⅤ fragment diagnostic for targeting.Conclusion The above-mentioned approaches can insure the successful α-GT gene knockout in mouse embryonic stem cells.It will provide information for α-GT gene knockout mouse;Mouse.
出处
《解剖学报》
CAS
CSCD
北大核心
2005年第2期173-176,共4页
Acta Anatomica Sinica
基金
重庆市攻关课题资助(2003 02008)