小鼠锌指蛋白基因Znf230条件基因打靶载体的构建

Conditional Targeting Vector of Mouse Zinc Finger Protein Gene(Znf230)

目的构建用于小鼠锌指蛋白基因Znf230条件基因打靶的载体,为产生Znf230基因敲除的小鼠模型准备条件。方法设计和合成引物,经PCR从小鼠的基因组中扩增出5′同源臂、3′同源臂及锚定序列,长度分别为4,4,1kb的片段,反向插入pBS载体的neo基因的两侧,从而构建小鼠Znf230条件基因打靶载体Znf230-pBS(5)。结果经过限制性内切酶及DNA测序鉴定,证实该条件基因打靶载体含有的同源序列与Genebank公布的基因序列一致,表明载体构建成功。结论PCR技术和定向克隆技术是构建条件基因打靶载体简单而可靠的方法。运用该技术可获得小鼠锌指蛋白基因Znf230的条件基因打靶载体。

Objective To construct a mouse zinc finger protein gene Znf230 conditional targeting vector. Methods Designing and synthesizing the specific primer, to amplify two homologous arms of 4 kb in 5＇ and 3＇ each and targeting sequence of 1 kb from mouse genome. Cloned them into two sides of neo gene in pBS vector. Results hZnf230-pBS was amplified by PCR with specific primer F1, R1 , F2, F3, F4 and R4, and the PCR products were 4, 4, 1, 1.1 kb length as expectant length of homologous arms and targeting sequence. Though REase digestion and sequencing analysis, the eDNA of the Znf230 was identified with the sequence in Genebank. It was also identified that the conditional targeting sequence was comprised exons Ill and its up and down stream 300 bp which was 100% as the Genebank sequence. Conclusion PCR and directional cloning is a simple and reliable method for construction targeting vector. The construction of mouse zinc finger protein gene Znf230 conditional targeting vector was obtained using this method.