scholarly journals Calcium phosphate transfection mammalian cells v1 (protocols.io.bthnnj5e)

protocols.io ◽  
2021 ◽  
Author(s):  
Karla LH
Keyword(s):  
Calcium Phosphate ◽  
Mammalian Cells ◽  
Calcium Phosphate Transfection

Phage Particle-Mediated Gene Transfer to Cultured Mammalian Cells

1982 ◽  
Vol 2 (6) ◽  
pp. 607-616
Author(s):  
Masahiro Ishiura ◽  
Susumu Hirose ◽  
Tsuyoshi Uchida ◽  
Yoshio Hamada ◽  
Yoshiaki Suzuki ◽  
...  
Keyword(s):  
Calcium Phosphate ◽  
Gene Transfer ◽  
Mammalian Cells ◽  
Phage Particle ◽  
High Efficiency ◽  
Linear Increase ◽  
Recombinant Phage ◽  
Optimal Values ◽  
Simplex Virus

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 10 5 phage particles per 10 6 cells without carrier DNA. An increase in the dosage of phage particles, up to at least 5 × 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.

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