tPA乳腺组织特异性表达载体构建及转基因小鼠建立

Construction of a Human Tissue-type Plasminogen Activator Tissue Specific Expression Vector and Establishment of Its Transgenic Mice

以 p KB、pc DNA 3和 p CPA为原始质粒 ,构建 BL G启动子调控的组织型纤溶酶原激活剂 (t PA)乳腺组织特异性表达载体 p BTA。以 Sal I酶切质粒 p BTA,回收 5 .45 kb基因片段 BL G- t PA- b GHpoly A,通过显微注射 ,导入昆明小鼠受精卵的雄原核内。共注 136 5枚 ,将存活的 10 5 3枚移植到 5 0只同期发情假孕母鼠的输卵管内。怀孕 2 4只 ,共产仔79只 ,上述仔鼠通过 PCR检测 ,结果 19只阳性。Southern blotting检测上述 19份 PCR阳性小鼠基因组 ,其中 4只为整合阳性 ,说明已获得了乳球蛋白启动子调控下的 t PA转基因小鼠 ,为 t PA在小鼠体内表达研究提供了必要条件。

Tissue-type plasminogen activator cDNA was cloned on downstream of the bovine beta-lactoglobulin promoter to form a expression vector pBTA. A 5.45kb fragment containing the tPA expression cassette was recovered and purified for microinjection. The microinjected ova were transferred to 50 pseudopregnant mouse recipients. 79 offspring were born. 19 of them were proved to be positive of gene integration by PCR, 4 mice were further confirmed to be transgenic by Southern hybridization.