Efficient DNA-mediated gene transfer into primary cultures of adult rat hepatocytes.
Efficient DNA-mediated gene transfer into primary cultures of adult rat hepatocytes.
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DOI：
10.1089/dna.1.1989.8.535
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发表时间：
1989-09
期刊：
影响因子：
--
通讯作者：
D. Pasco;J. Fagan
中科院分区：
文献类型：
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作者：
D. Pasco;J. Fagan
建立了一种高效、简便、重复性好的DNA介导的成年大鼠肝细胞原代培养的基因转移方法。与细胞孵育5小时后，在完整的培养液中形成磷酸钙-DNA沉淀物。然后洗掉未被吸收的沉淀物，并在40小时后测量基因表达。在最佳条件下，转入β-半乳糖苷酶(LacZ)基因的细胞中有高达20-25%的细胞表达这一活性，而融合到强启动子的氯霉素乙酰转移酶(CAT)基因的细胞表达CAT活性为每毫克蛋白10-14nmoles/min。根据转染率、CAT表达和细胞活力对5个条件进行了优化。(I)培养基组分：培养基中必须含有蛋白质，如胎牛血清或牛血清白蛋白。(2)细胞基质：组织培养塑料优于牛皮胶原蛋白和Matrigel。(3)细胞密度：0.5-1.0×10(6)个/60 mm培养皿优于较高密度。(4)接触磷酸钙-DNA的时间：5-8小时优于较短或较长时间。(5)肝细胞在开始转染期前维持培养的时间：2~3d较早。这一过程是成功的，报告基因连接到三个不同的真核启动子。其中包括一个含有细胞色素P450c基因的多环芳烃反应增强子(CYP1A1)的嵌合启动子，它被证明赋予CAT基因与天然P450c基因对多环芳烃的反应能力。这种转染法对于研究原代肝细胞培养中肝脏特异性基因的表达具有重要意义。
An efficient, simple, and reproducible DNA-mediated gene transfer procedure has been developed for primary cultures of adult rat hepatocytes. Calcium phosphate-DNA precipitate is formed in complete culture medium during 5 hr incubation with cells. Unabsorbed precipitate is then washed out, and 40 hr later gene expression is measured. Under optimal conditions, up to 20-25% of cells in cultures transfected with the beta-galactosidase (lacZ) gene stain positively for this activity, and cells transfected with the chloramphenicol acetyl transferase (CAT) gene, fused to a strong promoter, express CAT activities of 10-14 nmoles/min per mg protein. Five conditions were optimized based on transfection efficiency, CAT expression, and cell viability. (i) Medium composition: the presence of protein, such as fetal bovine serum or bovine serum albumin, in the medium was essential. (ii) Cell substratum: tissue culture plastic was superior to calf skin collagen and Matrigel. (iii) Cell density: 0.5-1.0 X 10(6) cells/60-mm dish were superior to higher densities. (iv) Duration of exposure to calcium phosphate-DNA: 5-8 hr was better than shorter or longer times. (v) Length of time hepatocytes were maintained in culture before initiating transfection: 2-3 days was superior to earlier times. This procedure was successful with reporter genes linked to three different eukaryotic promoters. These included a chimeric promoter containing the polycyclic aromatic hydrocarbon-responsive enhancer of the cytochrome P450c gene (CYP1A1), which was shown to confer upon the CAT gene responsiveness to polycyclic aromatic hydrocarbons comparable to that of the native P450c gene. This transfection procedure should be of considerable use for the study of liver-specific gene expression in primary hepatocyte cultures.