Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes.

Abstract

Coprecipitation of DNA with calcium phosphate is a commonly used method of gene transfer in mammalian cells. We have found that DNA forms a tight complex with Ca P. and that DNA in this complex is resistant to nucleases present in serum or added externally. Under optimal conditions, virtually all of the recipient mouse LtklAprt-cells take up Ca P.-DNA complexes, as determined by fluorescent dyes specific for DNA (4',6-diaminilo-2-phenylindol dihydrochloride) or for calcium salts (chlorotetracycline). However, only a small proportion of the cells have detectable Ca Pi-DNA complexes in the nucleus. Uptake of the Ca P-DNA complexes was highly dependent upon the pH at which the Ca P.-DNA complex was formed and upon the concentration of DNA in the complex.It has been known for many years that under certain conditions, eukaryotic cells can take up large amounts ofDNA and transport it into the cell nucleus (1, 2). This phenomenon has been exploited to achieve transformation of eukaryotic cells with both selectable and nonselectable genes (1, 2, ¶). Addition of polycations, such as polyornithine (3) or DEAE-dextran (3, 4), to the DNA solution or precipitation of the added DNA on the cell surface by calcium phosphate (5) have proven useful in increasing both the uptake ofthe DNA and the frequency ofcell transformation. Despite the large amount of high molecular weight DNA taken into cells in the presence of certain facilitators (3, 6), at-best only about one in 104 cells eventually becomes transformed (7,8). This has limited transfer to those genes for which there exists a good selective system and to certain cell lines, such as mouse L cells, that function as efficient recipients (8).The detailed mechanisms by which external DNA is integrated into a foreign chromosome and expressed in a foreign environment are as yet poorly understood. However, a large body of recent work (9, 10) has begun to elucidate the process. On the other hand, essentially nothing-is known about the way by which DNA molecules cross the cellular membrane and are transported to the nucleus. The studies described here were designed to study the quantitative aspects and the mechanisms of entry of DNA into cells after coprecipitation with Ca Pi. We have used the specific fluorescent dye 4',6-diaminilo-2-phenylindol dihydrochloride, (DAPI), which specifically stains double-stranded DNA (11), and chlorotetracycline, which stains complex salts ofCa (12). We show that virtually all recipient cells take up Ca Pf-DNA complexes, which appear in the cytoplasm in a characteristic structure, and that only a few ofthe cells have detectable Ca P,-DNA complexes in the nucleus. Additionally, DNA uptake was highly dependent on the pH at which the Ca Pi precipitate was formed and upon the concentration of DNA in that, precipitate. MATERIALS AND METHODSCell Culture. The murine cell line Ltk-Aprt-was cultured in minimum essential medium, a modification (GIBCO), sup-plemented with 10% (vol/vol) fetal calf serum. In most cases, cells were plated in 35-mm or 60-mm...

Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes

“…CPPs were prepared by mixing equal volumes of CaCl 2 (256 mM) and buffer A (50 mM HEPES, 3 mM Na 2 HPO 4 , pH 7.05,) as described previously (4) and were administrated dropwise to cells at 20% (v/v) for 4 h unless otherwise indicated. To visualize transfected DNA in cells, plasmid DNA (pDsRed, 25-50 g), SYBR Green, or propidium iodide (1 g/ml final concentration) was admixed with CaCl 2 solution (256 mM) before adding to buffer A as described previously (10). The CPP-DNA complex was then introduced to cells as in the case of CPPs alone.…”

New procedure for DNA transfection with polycation and dimethyl sulfoxide.

“…However, the calcium phosphate method has narrow optimal conditions for several factors, such as the pH of the buffer used, the amount of the DNAs, and the size of the calcium phosphate-DNA precipitate. Thus, the efficiency of transfection depends greatly on the conditions used (5,8,10). Furthermore, the mechanism for DNA uptake by the cell from calcium phosphate-DNA precipitate has not been fully elucidated (10).…”

How damaged is the biologically active subpopulation of transfected DNA?

“…Despite the usefulness of DNA transfection, very little is known about the pathway by which the biologically active molecules travel from outside the cell into the nucleus or about the status of the DNA once it arrives (19,27). Because the biologically active molecules constitute such a small portion of the total (commonly less than 1 part in 105), these questions are beyond the limits of routine biochemical investigation.…”