Phage particle-mediated gene transfer to cultured mammalian cells.
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 degrees 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 10(5) phage particles per 10(6) cells without carrier DNA. An increase in the dosage of phage particles, up to at least 5 x 10(7) phage particles per 100-mm dish, resulted in a linear increase in the number of transformants. Addition of carrier phage, up to 10(10) phage particles per dish, did not significantly affect the number of transformants.
Department of Cell Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444, Japan.
M
M
Ishiura
S
S
Hirose
T
T
Uchida
Y
Y
Hamada
Y
Y
Suzuki
Y
Y
Okada
D000327:Adsorption / Q000187:drug effects; D000818:Animals; D001435:Bacteriophages / Q000235:genetics* / Q000502:physiology; D002130:Calcium Phosphates; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002472:Cell Transformation, Viral; D011232:Chemical Precipitation; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D013395:Sucrose / Q000494:pharmacology; D013937:Thymidine Kinase / Q000235:genetics*; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D013395:Sucrose; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D013937:Thymidine Kinase; C485829:calcium phosphate, dibasic, anhydrous; D005990:Glycerol; D004121:Dimethyl Sulfoxide