A sensitive lacZ-based expression vector for analyzing transcriptional control elements in eukaryotic cells.
We have developed a eukaryotic expression vector that provides a rapid and sensitive measure of transcriptional activity modulated by general and tissue-specific regulatory motifs. The lacZ structural gene has been linked to the minimal promoter of the human liver/bone/kidney alkaline phosphatase gene. In addition, a trimerized cassette of the SV40 polyadenylation region has been placed 5' of this promoter to reduce plasmid-initiated transcripts extending through the lacZ gene that would contribute to background beta-galactosidase (beta-Gal) activity. By combining the weak promoter and the poly(A) cassette, only a very low level of lacZ activity is detected in the absence of additional regulatory sequences. Regulatory domains can be inserted into this vector via a unique Bam HI restriction site and their activity can be rapidly monitored in situ via a colorimetric 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Ga) staining protocol. Also, the activity of linked regulatory domains can be measured quantitatively by assaying beta-Gal levels in cell extracts. We show that derivatives of this vector can be used to monitor the activity of general and tissue-specific control elements and can be transactivated by a single transcription factor in cotransfection experiments.
Spear
B T
BT
Department of Microbiology & Immunology, University of Kentucky College of Medicine, Lexington 40536-0084, USA.
Longley
T
T
Moulder
S
S
Wang
S L
SL
Peterson
M L
ML
D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001842:Bone and Bones / Q000201:enzymology; D002460:Cell Line; D002522:Chlorocebus aethiops; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase