We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.

High-efficiency transformation of mammalian cells by plasmid DNA.

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.

https://www.pnas.org/content/pnas/84/21/7413.full.pdf

Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure

E. coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation). We have obtained 10(9) to 10(10) transformants/micrograms with strains LE392 and DH5 alpha, and plasmids pUC18 and pBR329. The process is highly dependent on two characteristics of the electrical pulse: the electric field strength and the pulse length (RC time constant). The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration. Most of the surviving cells are competent with up to 80% transformed at high DNA concentration. The mechanism does not appear to include binding of the DNA to the cells prior to entry. Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.

High efficiency transformation of E.coli by high voltage electroporation

https://www.cell.com/cell/pdf/0092-8674(87)90646-5.pdf

Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.

The ability to safely and efficiently transfer foreign DNA into cells is a fundamental goal in biotechnology. Toward this end, rapid advances have recently been made in our understanding of mechanisms for DNA stability and transport within cells. Current synthetic DNA delivery systems are versatile and safe, but substantially less efficient than viruses. Indeed, most current systems address only one of the obstacles to DNA delivery by enhancing DNA uptake. In fact, the effectiveness of gene expression is also dependent on several additional factors, including the release of intracellular DNA, stability of DNA in the cytoplasm, unpackaging of the DNA-vector complex, and the targeting of DNA to the nucleus. Delivery systems of the future must fully accommodate all these processes to effectively shepherd DNA across the plasma membrane, through the hostile intracellular environment, and into the nucleus.

Synthetic DNA delivery systems.

Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/- 3) transformants per 10(6) cells and per 12 micrograms DNA Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed According to this ' electroporation model' the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport

P. H. Hofschneider

Papers

Gene transfer into mouse lyoma cells by electroporation in high electric fields.