Oliveira Raniere R, Carvalho Daniela M de, Lisauskas Sharon, Mello Eduardo, Vianna Giovanni R, Dode Margot A N, Rumpf Rodolfo, Aragão Francisco J L, Rech Elíbio L
Laboratório de Transferência e Expressão de Genes, EMBRAPA Recursos Genéticos e Biotecnologia, Parque Estação Biológica, 70770-900 Brasília, DF, Brazil.
Genet Mol Res. 2005 Jun 30;4(2):185-96.
The development of an efficient transfection system in livestock cells is an important step towards investigating gene transfer and the functioning and production of transgenic animals. Important factors involved in cationic liposome mediated gene transfer were evaluated through in vitro transfection of bovine, caprine and ovine fibroblast cells. Transfection of plasmid DNA complexes of different commercially available liposomes (Lipofectamine, Lipofectin, Cellfectin and DMRIE-C; Gibco-BRL, USA) was evaluated utilizing the following parameters: DNA/liposome ratio, cell density, DNA conformation, and the effect of transfection time on the efficiency of bovine fibroblasts to express a reporter gene. The effects and concentrations of liposomes were also evaluated in caprine and ovine fibroblasts. Lipofectamine alone and Lipofectamine with Plus reagent induced high-frequency expression of beta-galactosidase and neo genes in all cells evaluated (47 and 88.3%, respectively). Regarding phenotype, chromosomal stability was similar in transfected and non-transfected cells. The parameters set in this study will establish a foundation for utilizing transfected fibroblast cells to generate transgenic animals through nuclear transfer technology and gene function studies.
在牲畜细胞中开发高效的转染系统是研究基因转移以及转基因动物的功能和生产的重要一步。通过对牛、山羊和绵羊成纤维细胞进行体外转染，评估了阳离子脂质体介导的基因转移所涉及的重要因素。利用以下参数评估了不同市售脂质体（Lipofectamine、Lipofectin、Cellfectin和DMRIE-C；美国Gibco-BRL）的质粒DNA复合物的转染情况：DNA/脂质体比例、细胞密度、DNA构象以及转染时间对牛成纤维细胞表达报告基因效率的影响。还评估了脂质体在山羊和绵羊成纤维细胞中的效果和浓度。单独的Lipofectamine以及Lipofectamine与Plus试剂在所有评估的细胞中均诱导了β-半乳糖苷酶和新霉素基因的高频表达（分别为47%和88.3%）。在表型方面，转染细胞和未转染细胞的染色体稳定性相似。本研究设定的参数将为利用转染的成纤维细胞通过核移植技术生成转基因动物和进行基因功能研究奠定基础。
