Aberrant cryptic responsiveness of the pCAT 3- and pGL3-promoter reporter vectors.
Transfection analyses are an informative method to assess the activity of specific promoter or enhancer elements in mammalian cells. Commercially available reporter vectors can be extremely useful investigative tools for such studies. This study reports that the pCAT 3- and pGL3-promoter vectors display cryptic responsiveness to androgens when they contain a DNA insert, while the empty vector, a commonly used negative control, is nonresponsive. Our studies initially aimed to characterize novel androgen-responsive DNA sequences in human genomic DNA through transactivational analyses. An isolated DNA fragment, designated ARC-3, contained three putative androgen response element "half-sites" and was androgen-responsive when cloned into the pCAT3-promoter vector. While we originally believed this to be a novel enhancer element, subsequent analyses of this clone revealed that this vector displays cryptic activity in the presence of an androgen. This was confirmed by cloning several unrelated DNA fragments that did not contain any known classic response elements into the pCAT3-promoter vector, all of which were found to be responsive. The empty vector (negative control) was again nonresponsive. The ARC-3 DNA fragment was also weakly responsive to stimulation when cloned into the pGL3-promotor vector, which is identical to the pCAT3-promoter vector, with the exception of an intron located 5' of the chloramphenicol acetyltransferase gene, and the reporter genes. This work demonstrates that both the pCAT3- and pGL3-promoter vectors are inappropriate to assess androgen-responsive enhancers and emphasizes the importance of the careful selection of reporter vectors and controls when conducting transactivational analysis.
Giannakis
G
G
Murdoch Childrens Research Institute, Austin and Repatriation Medical Center, Victoria, Australia.
Edmondson
S R
SR
Favaloro
J M
JM
Zajac
J D
JD
Greenland
K J
KJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry / Q000235:genetics*; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry; D004742:Enhancer Elements, Genetic; D004847:Epithelial Cells / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D005822:Genetic Vectors; D007438:Introns; D007668:Kidney / Q000166:cytology; D015741:Metribolone / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011944:Receptors, Androgen / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000235:genetics; D017384:Sequence Deletion; D015533:Transcriptional Activation; D014162:Transfection
D011944:Receptors, Androgen; D011965:Receptors, Glucocorticoid; D015741:Metribolone; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase