Evaluation of relative promoter strength in primary hepatocytes using optimized lipofection.
Evaluation of relative promoter strength in primary hepatocytes using optimized lipofection.
复制标题
使用优化的脂转染评估原代肝细胞中的相对启动子强度。
DOI：
10.1089/hum.1991.2.1-41
复制
发表时间：
1991
影响因子：
4.2
通讯作者：
Woo,SL
中科院分区：
文献类型：
--
作者：
Ponder,KP;Dunbar,RP;Wilson,DR;Darlington,GJ;Woo,SL
对于大多数表现在肝脏中的遗传缺陷，肝细胞中基因表达的最大化将是实现成功基因治疗的重要因素。一种快速、高效、无毒的将DNA转染入肝细胞的方法被用来直接比较各种细胞和病毒启动子的启动子强度。本文描述了使用带正电荷的脂质体Lipofectin转染5-10%原代肝细胞的条件。细胞不会被这种方法破坏，因为它们继续转录由肝脏特异性启动子控制的基因，并且可以在培养物中存活超过2周。我们发现巨细胞病毒、SRα和β-肌动蛋白启动子比SV 40、RSV、RNA聚合酶II、白蛋白、α1-抗胰蛋白酶或磷酸烯醇化羧激酶启动子更活跃。一个简单的TK启动子和TK启动子与多瘤增强子（MCI）几乎完全失活。这一信息将是有用的载体的建设，设计有效地表达基因在原代肝细胞的基因治疗的目的，虽然从这些启动子的表达的稳定性将需要证明在hepatocytesin体内。
For most genetic deficiencies manifested in the liver, maximization of gene expression in hepatocytes will be an important factor in achieving successful gene therapy. A rapid, highly efficient, and nontoxic method for transfecting DNA into hepatocytes was used to compare directly promoter strengths of various cellular and viral promoters. Conditions are described here for transfecting 5–10% of primary hepatocytes using the positively charged liposomes, Lipofectin. Cells are not damaged by this method as they continue to transcribe genes controlled by liver specific promoters and can survive for over 2 weeks in culture. We find that the cytomegalovirus, SRα, and β-actin promoters are more active than the SV40, RSV, RNA polymerase II, albumin, α1-antitrypsin, or phosphoenolpvriivatc carboxykinase promoters. A simple TK promoter and a TK promoter with the polyoma enhancer (MCI) were almost completely inactive. This information will be useful in the construction of vectors designed to express genes efficiently in primary hepatocytes for purposes of gene therapy, although the stability of expression from these promoters will need to be demonstrated in hepatocytesin vivo.