基因打靶载体选择标记盒的构建及表达

CONSTRUCTION OF SELECTION CASSETTE AND ITS EXPRESSION IN EUKARYOTIC CELLS

为构建山羊乳腺基因打靶载体的选择标记盒,将loxP位点分别引入正、反向引物,以含新霉素磷酸转移酶(neo)基因的质粒pTarget为模板,用高保真聚合酶链反应(PCR)扩增得loxP neo loxP选择标记盒。将PCR产物克隆到pGEM T载体后进行的序列分析结果显示,引入的loxP位点及扩增后的neo基因序列均是正确的。将此选择标记盒克隆到不含neo基因的真核表达载体ΔpTarget后转染COS 1细胞,经过G418筛选获得药物抗性细胞克隆。再将此选择标记盒克隆到山羊β 乳球蛋白基因打靶载体并转染山羊成纤维细胞,经过G418选择后也获得抗性细胞克隆。结果表明:克隆的loxP neo loxP选择标记盒可用于基因打靶载体的构建。

To generate a loxPneoloxP selection cassette for construction of goat mammary gland gene targeting vectors, loxP sites were introduced into both the forward and reverse primers and a plasmid pTarget containing neo gene was used as the template for high fidelity PCR amplification. Sequence analysis of the PCR product showed that both loxP sites and the amplified neo gene were correct. The loxPneoloxP cassette was subcloned into an eukaryotic expression vectorΔpTarget without neo gene and a goat mammary gland gene targeting vector for transfection of COS1 cell line and goat primary fibroblast cells. After two weeks selection, G418resistant cell clones were obtained. These data indicate that the amplified loxPneoloxP selection cassette is usable for construction of gene targeting vectors.