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首页> 外文期刊>Molecular and Cellular Biology >Phage Particle-Mediated Gene Transfer to Cultured Mammalian Cells
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Phage Particle-Mediated Gene Transfer to Cultured Mammalian Cells

机译：噬菌体颗粒介导的基因转移到培养的哺乳动物细胞

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摘要

Recombinant phage particles carrying the thymidine kinase (TK) gene of herpes simplex virus type 1, coprecipitated with calcium phosphate, efficiently transformed mouse Ltk? cells to the TK+ phenotype. The conditions necessary to achieve high efficiency of transfer of the TK gene by phage particle-mediated gene transfer were investigated. Of the parameters examined, the pH of the buffer used for coprecipitation of phage particles with calcium phosphate, the length of time of coprecipitation, and the length of the adsorption period were found to alter the transfer efficiency significantly. The optimal pH was 6.87 at 25°C. The other optimal values for these parameters were as follows: coprecipitation time, 7 to 20 min; adsorption time, 18 to 30 h. Treatment with dimethyl sulfoxide, glycerol, or sucrose did not enhance gene transfer. The optimal conditions yielded about 1 transformant per 105 phage particles per 106 cells without carrier DNA. An increase in the dosage of phage particles, up to at least 5 × 107 phage particles per 100-mm dish, resulted in a linear increase in the number of transformants. Addition of carrier phage, up to 1010 phage particles per dish, did not significantly affect the number of transformants.
机译：携带与磷酸钙共沉淀的1型单纯疱疹病毒胸苷激酶（TK）基因的重组噬菌体颗粒有效地将小鼠Ltk ？细胞转化为TK + 表型。研究了通过噬菌体颗粒介导的基因转移实现TK基因高效转移的必要条件。在所检查的参数中，发现用于噬菌体颗粒与磷酸钙共沉淀的缓冲液的pH，共沉淀的时间长度和吸附时间的长度显着改变了转移效率。最佳pH在25°C为6.87。这些参数的其他最佳值如下：共沉淀时间为7至20分钟;吸附时间为18至30小时。用二甲基亚砜，甘油或蔗糖处理不会增强基因转移。在没有载体DNA的条件下，每10 6 细胞的最佳条件是每10 5 噬菌体颗粒产生约1个转化子。每100毫米培养皿中噬菌体颗粒剂量的增加至少达到5×10 7 噬菌体颗粒，导致转化体数量线性增加。每个培养皿中最多添加10 10 噬菌体颗粒的载体噬菌体并没有显着影响转化体的数量。

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