Tartrate-resistant acid phosphatase gene expression as a facile reporter gene for screening transfection efficiency in mammalian cell cultures.
The efficiency of DNA transfection into mammalian cell cultures has been monitored using a variety of reporter assays. However, the common procedures are expensive, time-consuming and usually cannot identify the transfected cell population directly. In the present communication we describe a simple, inexpensive and efficient method to directly identify DNA transfection in mammalian cells using tartrate-resistant acid phosphatase (TRAP) gene expression. The method involves the transfection of a plasmid (pCT3), which contains TRAP cDNA driven by a CMV promoter, into mammalian cells. The cells can then be stained for TRAP activity, and the transfection efficiency can be determined by simply counting the positively transfected cells in a defined area with a microscope. This method permits screening of mammalian cells for transfection efficiency in multi-well plates. After waiting 30-40 minutes to allow the TRAP assay to saturate, wells can be scored in 1-2 minutes with little difficulty in detecting the transfected cells.
Reddy
S V
SV
Department of Medicine/Hematology, University of Texas Health Science Center, San Antonio.
Takahashi
S
S
Haipek
C
C
Chirgwin
J M
JM
Roodman
G D
GD
D000135:Acid Phosphatase / Q000032:analysis / Q000235:genetics*; D000818:Animals; D002130:Calcium Phosphates; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D011401:Promoter Regions, Genetic; D013194:Staining and Labeling; D013644:Tartrates / Q000494:pharmacology*; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D002130:Calcium Phosphates; D018076:DNA, Complementary; D013644:Tartrates; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D000135:Acid Phosphatase; D001616:beta-Galactosidase; C485829:calcium phosphate, dibasic, anhydrous; C029768:tartaric acid