Wilson S P, Liu F, Wilson R E, Housley P R
Department of Pharmacology, University of South Carolina School of Medicine, Columbia 29208, USA.
Anal Biochem. 1995 Apr 10;226(2):212-20. doi: 10.1006/abio.1995.1216.
Optimal conditions for formation of calcium phosphate-DNA precipitates and for chromaffin cell transfection by the calcium phosphate method were examined. A relationship was observed between turbidity of calcium phosphate solutions and the ability of calcium phosphate-DNA mixtures to give efficient transfection of bovine chromaffin cells. Under optimal conditions up to 35% of chromaffin cells in cultures transfected with plasmid DNA encoding human proenkephalin or Escherichia coli beta-galactosidase expressed the respective proteins. Important factors for transfection were the pH (6.95) and buffer employed for calcium phosphate-DNA precipitate formation, the amount and type of DNA, and the absence of serum in the cultures. Additionally, phosphate and calcium concentrations in the culture medium during incubation of cells with DNA are critical. Optimal conditions for transfection of chromaffin cells were also useful for transfection of clonal BSC-40 cells, an African green monkey kidney cell line. These results suggest that the optimal conditions described here for chromaffin cells may have broad applicability to other cell types. In addition, the results suggest that it is possible to optimize the solutions used for transfection conditions by monitoring calcium phosphate formation.
研究了磷酸钙-DNA沉淀物形成以及通过磷酸钙法转染嗜铬细胞的最佳条件。观察到磷酸钙溶液的浊度与磷酸钙-DNA混合物有效转染牛嗜铬细胞的能力之间存在关联。在最佳条件下，用编码人前脑啡肽或大肠杆菌β-半乳糖苷酶的质粒DNA转染的培养物中，高达35%的嗜铬细胞表达了相应的蛋白质。转染的重要因素包括用于形成磷酸钙-DNA沉淀物的pH值（6.95）和缓冲液、DNA的量和类型以及培养物中无血清。此外，细胞与DNA孵育期间培养基中的磷酸盐和钙浓度至关重要。嗜铬细胞转染的最佳条件对克隆的BSC-40细胞（一种非洲绿猴肾细胞系）的转染也很有用。这些结果表明，此处描述的嗜铬细胞最佳条件可能广泛适用于其他细胞类型。此外，结果表明通过监测磷酸钙的形成来优化用于转染条件的溶液是可能的。
