A Sensitive lacZ -Based Expression Vector for Analyzing Transcriptional Control Elements in Eukaryotic Cells

Abstract

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 β-galactosidase (β-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-β-D-galactoside (X-Gal) staining protocol. Also, the activity of linked regulatory domains can be measured quantitatively by assaying β-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.