Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane.
The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in medium containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual human chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8 X 10(-4)-3 X 10(-3). This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.
Tao
W
W
Wilkinson
J
J
Stanbridge
E J
EJ
Berns
M W
MW
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D006822:Hybrid Cells / Q000166:cytology; D007041:Hypoxanthine Phosphoribosyltransferase / Q000172:deficiency / Q000235:genetics; D007621:Karyotyping; D007834:Lasers; D051379:Mice; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D014170:Transformation, Genetic
D007041:Hypoxanthine Phosphoribosyltransferase