Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells.

Abstract

Strontium ion formed DNA-phosphate precipitates analogous to those formed by calcium but lacking the lethal and differentiation-inducing effects of calcium on many epithelial cell types in primary culture. Human primary bronchial epithelial cells were transiently and stably transfected by using strontium phosphate; the frequency of stable transformation with a plasmid carrying the simian virus 40 large-T-antigen gene was greater than l0-4.Calcium phosphate transfection has been widely used to introduce cloned and genomic DNAs and RNAs into cultured cell lines and intact animals (2-4, 6, 8, 12, 26, 30 (14) on collagen-fibronectin-coated 100-mm dishes in semidefined serum-free mediium LHC-9 (pH 7.3 to 7.4) in a 4.5% CO2 humidified incubator. At 18 h prior to transfection, the incubator CO2 level was checked and cells were passaged by using 0.02% trypsin-0.02% The 2 M SrCl2 solution was prepared in distilled water and filter sterilized. Sources were BDH, Poole, England (0.05% Ca; distributed by Gallard-Schlesinger, Carle Place, N.Y.) and Johnson Matthey Chemicals, Royston, Hertfordshire, England (Puratronic; 0.0001% Ca; distributed by AESAR, Seabrook, N.H.). It was important to rinse filters with distilled water prior to filtering these solutions to avoid subsequent aggregation of precipitate. A 2x HEPES balanced salt solution (HBS; 2x HBS is 8.18 g of NaCI-5.95 g of HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]4.2 g of Na2HPO4 -7H20-distilled water to 500 ml) (5, 30) was pH adjusted to 7.7 to 7.9 with NaOH, filter sterilized, and stored at 4°C. For calcium phosphate, 2 M CaC12 (BDH) was used and 2x HBS was adjusted to pH 7.08. Glycerol was 15% (wt/vol) in HBS or 11.8% (vol/vol); the solution was filter sterilized and stored at 4°C.To prepare precipitates, solutions were warmed to room temperature. For a 25-cm2 flask, 5 ,zg of DNA in x ,ul of solution was transfected by consecutively mixing 220 -x il of sterile distilled water, 31 ,ul of 2 M SrC12, and x ,ul of DNA (cesium chloride banded and supercoiled) in a sterile nonwettable plastic tube (Falcon 2059; Becton Dickinson Labware, Oxnard, Calif.). This solution was added dropwise to 250 ,ul of 2x HBS, with mixing between drops to prevent high local concentrations of phosphate. Mixing was done by adding the solution to the depression created in the 2x HBS by a nitrogen gas stream. As the mixture stood, the precipitate increased in size, first forming small dustlike threads and then forming progressively larger networks. The size of the precipitates was monitored by phase-contrast microscopy immediately after successive samples were transferred to a slide without a cover slip. At the stage of fine dust (5 to 10 min), the precipitates were pipetted into the flasks. The efficacy of transfection could be predicted from the quality of the precipitate. After 90 min (NHBE), 2 h (CV-1), or 3 h (NIH 3T3), the cells were rinsed twice with serum-free medium at 37°C, incubated with 1.5 ml of 15% glycerol for 30 s at room temperature (NHBE), 3 min at 37°C (CV-1...

Enhancement of calcium phosphate-mediated transfection by inclusion of adenovirus in coprecipitates

“…Moreover, because of the limited effectiveness, DNA:CaPi coprecipitates have been applied to cells for long incubation times in simple NaCl solutions that have led to cell toxicity. 22,26,27 For example, a standard protocol involves treating cells with DNA:CaPi coprecipitates in Hepes-buffered saline (pH 7.1) for 16 h. 15 We also found that with long exposure times CaPi could produce significant cell toxicity. In earlier work, we found that CaPi coprecipitates were relatively effective at binding to cells, and we were able to use this property to enhance markedly delivery of adenovirus to cells that were normally resistant to infection.…”

Tolerance of high levels of wild-type p53 in transformed epithelial cells dependent on auto-regulation by mdm-2

“…doses have previously been shown to delay the induction of mdm-2 expression in murine C127 cells (Perry et al, 1993), and u.v. fails to induce mdm-2 expression in murine keratinocytes in vivo (Burden et al, 1996). Thus repression of mdm-2 expression may contribute to the reportedly higher e cacy of u.v.…”

“…Cells growing in 90 mm dishes were transfected by strontium phosphate co-precipitation (Brash et al, 1987). 20 mg of the p53 dependent reporter plasmid pRGCDfoslacZ or pDfos-lacZ control vector (Frebourg et al, 1992) (a gift from S Friend) was co-transfected with 2 mg of either pSV 2 neo (Southern and Berg, 1982) or pC53-SCX3 (Baker et al, 1990), which encodes the temperature sensitive ala 143 mutant of human p53 (Zhang et al, 1994) and the neo gene.…”

Characterisation of human thyroid epithelial cells immortalised in vitro by simian virus 40 DNA transfection

“…Transfection was performed by the method of strontium phosphate coprecipitation (Brash et al, 1987). Six hours before transfection, the medium was replaced with 5 ml of warm SF-12 medium (Flow Labs) with 10% fetal calf serum.…”

“…Most success has been achieved with human mammary epithelium and keratinocytes (Brown & Gallimore, 1987;Taylor-Papadimitrou et al, 1982) transformed by SV40. Transformation of human brochial epithelium with SV40 large T (Brash et al, 1987;Reddel et al, 1988), of human uroepithelial cells (Christian et al, 1987) and fetal colonic epithelial cells (Berry et al, 1988) with SV40, and human thyroid epithelium with SV40 early region by electroporation (Whitley et al, 1987) has also been reported. Recently infection with a retrovirus carrying the adenovirus EIA gene has been used to generate a human thyroid cell line (Cone et al, 1988).…”