Antin P B, Mar J H, Ordahl C P
Dept. of Anatomy, University of California, San Francisco 94143.
Biotechniques. 1988 Jul-Aug;6(7):640-2, 645-9.
Transfection experiments involving mixed cell cultures pose special problems because of differential uptake and expression of DNA by different cell types. In order to evaluate the expression of transfected DNA in primary cultures of embryonic myocardial cells, we have used immunofluorescence microscopy to analyze expression of chloramphenicol acetyltransferase (CAT) on a single cell basis. The results show that myocardial cells take up and express transfected DNA approximately 7 times less efficiently than non-myocardial cells. The immunofluorescence analysis allows the biochemical level of CAT activity expressed under control of different transcriptional promoters to be compared on a cell type by cell type basis. Thus, the relative transcriptional strength of two promoters can be estimated for a specific cell type within a heterogeneous population of cells.
由于不同细胞类型对DNA的摄取和表达存在差异，涉及混合细胞培养的转染实验存在特殊问题。为了评估转染DNA在胚胎心肌细胞原代培养物中的表达，我们使用免疫荧光显微镜在单细胞水平上分析氯霉素乙酰转移酶（CAT）的表达。结果表明，心肌细胞摄取和表达转染DNA的效率比非心肌细胞低约7倍。免疫荧光分析能够在细胞类型逐个比较的基础上，对在不同转录启动子控制下表达的CAT活性的生化水平进行比较。因此，可以在异质细胞群体中针对特定细胞类型估计两个启动子的相对转录强度。
