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Mol Cell Biol. 1982 June; 2(6): 607-616

Phage Particle-Mediated Gene Transfer to Cultured Mammalian Cells

¹ Department of Cell Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444, Japan
² Department of Developmental Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444, Japan
³ Department of Animal Virology, Research Institute for Microbial Diseases, Yamada-oka, Suita 565, Japan

ABSTRACT

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 x 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.

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