Expression of transfected DNA by primary murine keratinocytes.
Primary murine keratinocytes can be maintained in culture for extended periods in a proliferative, basal cell state under conditions of reduced extracellular Ca2+. In response to increased Ca2+ concentrations, the cells undergo a well-defined program of terminal differentiation, thus serving as a convenient model in which to study the genes involved in regulating this and possibly other differentiation cascades by DNA-mediated gene transfer. However, because of their sensitivity to increased Ca2+ concentrations, the introduction of exogenous genomic DNA into primary keratinocytes by conventional methods is problematic. We have optimized the calcium phosphate DNA transfection procedure by introducing conditions that reduce the potency of Ca2+ as a differentiation signal. Primary epidermal cells were transfected with pSV2CAT, a plasmid that codes for the enzyme chloramphenicol acetyltransferase CAT. Enzyme activity was measured in cell extracts under varying transfection conditions. When the K+ concentration of the medium used for transfection by calcium phosphate precipitation is reduced from 6.5 to 0.01 mM, CAT activity following transfection increases 2-3 times. Exposure to the DNA precipitate for 2-4 h is optimal. By the use of fibroblast conditioned medium following transfection, enzyme activity can be detected in cell extracts for at least 21 d, suggesting that the exogenous gene is integrated. The low K+/Ca2+ transfection method is more effective than SrCl2 used as an alternative for CaCl2 in Ca2+ sensitive cells. Low K+ medium enhances cell survival for Ca2+ mediated transfection but also appears to have a beneficial effect on DNA uptake or expression.
Harper
J R
JR
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892.
Greenhalgh
D A
DA
Yuspa
S H
SH
D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase; D004247:DNA / Q000502:physiology*; D004817:Epidermis / Q000378:metabolism*; D005347:Fibroblasts / Q000502:physiology; D007633:Keratins; D051379:Mice; D008807:Mice, Inbred BALB C; D011188:Potassium / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D007633:Keratins; D004247:DNA; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D011188:Potassium; D002118:Calcium