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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
3
|
| pubmed:dateCreated |
1995-11-30
|
| pubmed:abstractText |
Valid comparisons of gene promoter activities between different cell lines, and within a cell line, critically depend on accurate measurements of the number of genes introduced into the nuclei of cells. We have developed a simple method that allows direct and accurate quantitation of transfected plasmid DNA in cultured cells. The transfected DNA present in nuclei is copurified with genomic DNA without using phenol/chloroform extractions. DNA is amplified by PCR, and the amount of transfected DNA is read directly from a standard curve. By using the procedures described in this report, we have studied the relative expression of the Escherichia coli beta-galactosidase gene, driven by the wild-type Mo-MuLV LTR, in Rat-1 fibroblasts, FBJ v-fos-transformed Rat-1 (1302), and a revertant of v-fos-transformant (EMS-1-19) cell lines. The relative levels of expression of the transgene at 22 hr post-transfection in these three cell lines were 1:4:1, respectively, and at 48 hr post-transfection the respective ratios were 1:10.6:4. These results have significant implications for the use of cotransfected internal control plasmids to normalize data from transient transfection experiments to study promoter activities among different cell lines.
|
| pubmed:grant | |
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/DNA,
http://linkedlifedata.com/resource/pubmed/chemical/Endopeptidase K,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Serine Endopeptidases,
http://linkedlifedata.com/resource/pubmed/chemical/beta-Galactosidase
|
| pubmed:status |
MEDLINE
|
| pubmed:month |
Dec
|
| pubmed:issn |
1054-9803
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:volume |
4
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
145-53
|
| pubmed:dateRevised |
2008-11-21
|
| pubmed:meshHeading |
pubmed-meshheading:7580898-Animals,
pubmed-meshheading:7580898-Cell Line,
pubmed-meshheading:7580898-Cell Nucleus,
pubmed-meshheading:7580898-Cell Transformation, Neoplastic,
pubmed-meshheading:7580898-DNA,
pubmed-meshheading:7580898-Electroporation,
pubmed-meshheading:7580898-Endopeptidase K,
pubmed-meshheading:7580898-Gene Expression,
pubmed-meshheading:7580898-Genes, fos,
pubmed-meshheading:7580898-Kinetics,
pubmed-meshheading:7580898-Moloney murine leukemia virus,
pubmed-meshheading:7580898-Plasmids,
pubmed-meshheading:7580898-Polymerase Chain Reaction,
pubmed-meshheading:7580898-Promoter Regions, Genetic,
pubmed-meshheading:7580898-Rats,
pubmed-meshheading:7580898-Recombinant Proteins,
pubmed-meshheading:7580898-Repetitive Sequences, Nucleic Acid,
pubmed-meshheading:7580898-Serine Endopeptidases,
pubmed-meshheading:7580898-Transfection,
pubmed-meshheading:7580898-beta-Galactosidase
|
| pubmed:year |
1994
|
| pubmed:articleTitle |
Direct gene quantitation by PCR reveals differential accumulation of ectopic enzyme in rat-1 cells, v-fos transformants, and revertants.
|
| pubmed:affiliation |
Division of Toxicology, Whitaker College of Health Science and Technology, Massachusetts Institute of Technology, Cambridge 02139, USA.
|
| pubmed:publicationType |
Journal Article,
Comparative Study,
Research Support, U.S. Gov't, P.H.S.
|