Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency

Journal Article · Wed Sep 01 04:00:00 EDT 1982 · Mol. Cell. Biol.; (United States)

DOI:https://doi.org/10.1128/MCB.2.9.1145· OSTI ID:5964000

Shen, Y M; Hirschhorn, R R; Mercer, W E; Surmacz, E; Tsutsui, Y; Soprano, K J; Baserga, R

The authors have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (--10/sup 4/ base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. They compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in tansfectability of different cell lines. In the laboratory, the cell line that had the highest efficiency of transfection was tk/sup -/ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. The experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.

• Research Organization: Departments of Microbiology and Pathology and Fels Research Institute, Temple Univ. School of Medicine, Philadelphia, PA 19140
• OSTI ID: 5964000
• Journal Information: Mol. Cell. Biol.; (United States), Journal Name: Mol. Cell. Biol.; (United States) Vol. 2:9; ISSN MCEBD
• Country of Publication: United States
• Language: English

Related Subjects

550400* -- Genetics
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
AZINES
BODY
CELL CONSTITUENTS
CELL CULTURES
DISEASES
DNA
ENZYME ACTIVITY
ENZYMES
GENE MUTATIONS
GENES
GENETIC ENGINEERING
GENOTYPE
HAMSTERS
HERPES SIMPLEX
HETEROCYCLIC COMPOUNDS
IMMUNOASSAY
IMMUNOLOGY
INFECTIOUS DISEASES
ISOTOPE APPLICATIONS
KIDNEYS
MAMMALS
MICROORGANISMS
MOLECULAR WEIGHT
MUTATIONS
NUCLEIC ACIDS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PARASITES
PHENOTYPE
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PLASMIDS
PYRIMIDINES
RADIOASSAY
RADIOIMMUNOASSAY
RADIOIMMUNOLOGY
RECOMBINANT DNA
RIBOSIDES
RODENTS
SIMIAN VIRUS
SKIN DISEASES
TEMPERATURE EFFECTS
THYMIDINE
TRACER TECHNIQUES
TRANSFERASES
VERTEBRATES
VIRAL DISEASES
VIRUSES