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 cell...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1987-06, Vol.84 (12), p.4180-4184
Main Authors: Tao, Wen, Wilkinson, Joyce, Stanbridge, Eric J., Berns, Michael W.
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Biotechnology
Cell Line
Cell lines
Cell Membrane - ultrastructure
Cell membranes
Chromosomes
Cultured cells
DNA
fibrosarcoma
Fundamental and applied biological sciences. Psychology
gene transfer
Genes
Genetic engineering
Genetic technics
Genetic Vectors
Humans
Hybrid Cells - cytology
Hypoxanthine Phosphoribosyltransferase - deficiency
Hypoxanthine Phosphoribosyltransferase - genetics
Karyotyping
L cells
Lasers
Methods. Procedures. Technologies
Mice
Nucleic Acid Hybridization
Plasmids
transformation
Transformation, Genetic
Transformed cell line
Vectors (cloning, transfer, expression). Insertion sequences and transposons
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Summary: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 × 10-4-3 × 10-3. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.84.12.4180