人类α-actin启动子真核表达载体的构建及应用  

Construction of Eukaryotic Expression Vector Containing Human Cardiac α-actin Promoter and It's Application

作　　者：杨玉艾[1] 孙永科[1] 华进联[1] 窦忠英[1] 

机构地区：[1]西北农林科技大学陕西省干细胞工程技术中心,杨凌712100

出　　处：《畜牧兽医学报》2006年第11期1093-1098,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家"863"(2002AA216161);国家自然科学基金(30200137)

摘　　要：利用PCR技术克隆了人类α-actin基因的启动子（约450bp），尝试用去掉启动子的pEGFP—N1作为框架结构，成功构建了真核表达载体pEGFP-N1-α-actin—P。应用Lipofectamine 2000将所构建的pEGFP-N1-α-actin-P转染入小鼠ES细胞。通过比较，发现在本实验室条件下，质粒DNA浓度以3．0～5．0μg／mL，转染时间约在2～3h最佳。200μg／mL的G418较适宜于靶细胞的转染与筛选。得到表达心肌a—actin和GFP的小鼠ES细胞，基本维持小鼠ES细胞的形态。PCNA染色结果表明，转染后的小鼠ES细胞具有增殖能力。α—actin抗体免疫组化染色结果表明，转染后细胞表达α-actin和GFP，揭示构建的真核表达载体pEGFP-N1-α-actin-P转染小鼠ES细胞，可能促进其向心肌细胞分化并对其筛选。Human cardiac α-actin promoter obtained from the human heart genome by polymerase chain reaction（PCR） was ligated into the same fragment of pEGFP-N1（moved CMV promoter by AseⅠ/Bgl Ⅱ）. The constructed expression vector, named α-actin-pEGFP-N1 which contains neo＇ gene and GFP gene as positive selection markers, was transferred into mice ES cells by Lipofectamine 2000. It＇s found that DNA concentration should be 3.0-5.0μg/mL, transfer time was 2-3h, 200μg/mL G418 was used as perfect working concentration. Fluorescent observation and immunocytochemistry assay （PCNA and antibody of α-actin） demonstrated the transfected cells show high levels of α-actin and GFP expression, basically maitain its natural characteristic compared with wild mice ES cells. The results suggest that human cardiac α-actin promoter may be useful in the study of mice ES cells specific differentiation into cardiomyocytes.

关 键 词：人类ractin启动子 真核表达载体 小鼠ES细胞 心肌细胞 

分 类 号：Q813[生物学—生物工程]