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 genera l and tissue-specific regulatory moths. The lacZ structural gene has b een linked to the minimal promoter of the human liver/bone/kidney alka line phosphatase gene. In addition, a trimerized cassette of the SV40 polyadenylation region has been placed 5' of this promoter to reduce p lasmid-initiated transcripts extending through the lacZ gene that woul d 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 regula tory sequences. Regulatory domains can be inserted into this vector vi a a unique Barn HI restriction site and their activity can be rapidly monitored itt situ via a colorimetric 5-bromo-4-chloro-3-indolyl-beta- D-galactoside (X-Gal) 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 i n cotransfection experiments.