Rapid characterization of growth-arrest genes in transient transfection assays
We developed a rapid assay for identifying growth-arrest genes to facilitate studies of cell cycle regulation. A7r5 vascular smooth muscle cells were transiently transfected with two plasmids: (i) a pMSV beta Gal reporter construct expressing beta-galactosidase (beta-gal) under transcriptional contr...
| Veröffentlicht in: | BioTechniques. - 1988. - 18(1995), 3 vom: 06. März, Seite 434-6, 438, 440-2 |
|---|---|
| 1. Verfasser: | |
| Weitere Verfasser: | , |
| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
1995
|
| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. DNA 9007-49-2 Cyclin-Dependent Kinases EC 2.7.11.22 Bromodeoxyuridine G34N38R2N1 |
| Zusammenfassung: | We developed a rapid assay for identifying growth-arrest genes to facilitate studies of cell cycle regulation. A7r5 vascular smooth muscle cells were transiently transfected with two plasmids: (i) a pMSV beta Gal reporter construct expressing beta-galactosidase (beta-gal) under transcriptional control of the murine sarcoma virus long terminal repeat; and (ii) a eukaryotic expression vector driving transcription of a potential growth inhibitory c-DNA under control of the cytomegalovirus promoter/enhancer. Twenty-four hours after transfection, cellular DNA was labeled for an additional 24 h with 5-bromo-2-deoxyuridine (BrdU) to label cellular DNA. After fixation, transfected cells were identified by histochemical staining with a beta-gal substrate, 6-chloro-3-indolyl-beta-D-galactopyranoside (i.e., Red-Gal). Transfected cells (beta-gal-positive) that traversed S phase (i.e., DNA synthesis) were quantified by indirect immunocytochemical staining for BrdU. Since autoradiography was not required to score for DNA synthesis, the length of experiments was much shorter than previously described growth-arrest assays performed with transiently transfected cells. Experiments with two growth-arrest genes, p53 and the p21 cyclin-dependent kinase inhibitor, demonstrated the utility of this assay |
|---|---|
| Beschreibung: | Date Completed 20.07.1995 Date Revised 21.11.2013 published: Print Citation Status MEDLINE |
| ISSN: | 1940-9818 |