人CCL20基因打靶载体的构建与鉴定

Construction and Identification of Human CCL20 Gene Targeting Vector

目的:构建针对人CC亚族趋化因子配体20(CC chemokine ligand20,CCL20)基因外显子2的置换型打靶载体。方法:设计和合成引物,利用长距离聚合酶链反应(polymerase chain reaction,PCR)从永生化人角质形成细胞系(HaCaT)基因组DNA中克隆出CCL20基因组DNA片段,再以CCL20基因为模板扩增出长度为1969bp的同源短臂和2356bp的同源长臂,分别插入ploxP载体的Neo基因上游和下游,构建针对人CCL20基因外显子2的置换型打靶载体(ploxP-hCCL20)。将打靶载体线性化并以电穿孔方法转移入HaCaT,观察克隆的形成。结果:经过PCR、限制性内切酶鉴定及DNA序列测定,证实该载体的两条同源臂包含人CCL20基因的外显子1、3、4及其邻近的部分内含子。结论:ploxP-hCCL20载体构建成功,增加Neo基因下游同源臂长度的策略有可能提高细胞基因敲除的同源重组率。

Objective： To construct a replacement targeting vector for exon 2 of human CC chemokine ligand 20 （hCCL20）. Methods： The short arm （1969 bp） and long arm （2356 bp） for human CCL20 gene targeting vector were isolated from the genomic DNA of human immortal skin keratinocyte line （HaCaT） by long polymerase chain reaction （PCR）. These fragments were cloned into ploxP vector at both sides of the neomycin resistance gene, respectively. Then, the constructed gene targeting vector against human CCL20, ploxP-hCCL20, was identified by PCR, restricted endonucleases digestion and DNA sequencing. The EcoR I-linearized targeting vector was electroporated into HaCaT. Results： The two homologous arms including exonl,3,4 were cloned into ploxP vector and identified by PCR, restricted endonucleases digestion and sequence analysis. Conclusion： The replacement targeting vector for human CCL20 gene has been successfully reconstructed.