Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Expression of transfected DNA by primary murine keratinocytes. 245 57

Osteocalcin is a major noncollagenous protein of bone regulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and is believed to be expressed only by differentiated osteoblasts. We introduced a 3.9-kilobase human osteocalcin gene promoter (hOCP)-chloramphenicol acetyltransferase (CAT) fusion gene into the germ line of mice. Examination of tissue extracts from these transgenic mice demonstrated that the expression of CAT was restricted to bone-associated tissues and the brain. Immunohistochemical staining of femur tissue sections using CAT antibodies localized the production of CAT protein to osteoblasts and maturing chondrocytes. Previous studies via transient transfection into osteoblast-like cells have identified a vitamin D response element approximately 500 basepairs up-stream of the hOCP capable of mediating 1,25-(OH)2D3 induction. As a consequence, regulation of the transgene was examined in homozygous transgenic lines for sensitivity to 1,25-(OH)2D3. Hormonal deficiency was created using a low calcium diet supplemented with 0.8% SrCl2 for 7 days and was restored in experimental mice by injection of 25 ng 1,25-(OH)2D3/day, ip, for 3 days. The low vitamin D3 diet decreased CAT activity several-fold in extracts from calvaria, femur, and brain compared to that in mice maintained on a normal diet, while 1,25-(OH)2D3 supplementation restored and enhanced CAT activity over control values. These data demonstrate that hOCP is sufficient to direct osteoblast-specific 1,25-(OH)2D3-sensitive gene expression in mice in addition to the unexpected regulatable expression in brain tissue.
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PMID:The human osteocalcin promoter directs bone-specific vitamin D-regulatable gene expression in transgenic mice. 848 81