Intestine-specific expression of green fluorescent protein-tagged cathepsin B: proof-of-principle experiments.
We hypothesized that tissue-specific expression of cathepsin B-enhanced green fluorescent protein (CB-EGFP) can be driven by the A33-antigen promoter that contains positive cis-regulatory elements, including caudal-related homeobox (CDX) binding sites. The intestine-specific transcription factor Cdx1 is crucial for A33-antigen promoter activation and could thereby induce expression of CB-EGFP. This concept was tested by construction of the vector pA33-CathB-EGFP encoding CB-EGFP downstream of the A33-antigen promoter. Its Cdx1 dependence, as an indication of its intestine-specific expression, was tested in Cdx1-negative CHO-K1 cells. Cdx1 expression was achieved upon transfection with pCdx1-DsRed-Express and was indicated by red fluorescence of the simultaneously translated reporter protein. Immunolabeling with Cdx1-specific antibodies showed correct targeting of the transcription factor to its point of action in nuclei of transfected cells. Co-transfection experiments with plasmids pA33-CathB-EGFP and pCdx1-DsRed-Express confirmed the hypothesis that Cdx1 indeed activates CB-EGFP expression in a manner dependent on the A33-antigen promoter. Co-localization with compartment-specific markers and subcellular fractionation confirmed CB-EGFP trafficking along the expected route to endolysosomal compartments. Hence, the A33-antigen promoter represents a potent tool for induction of Cdx1-dependent CB-EGFP expression in vitro. Our proof-of-principle studies confirm the suitability of this approach in visualizing protease transport in Cdx1-positive tissues of the gastrointestinal tract.
Mayer
Kristina
K
School of Engineering and Science, Jacobs University Bremen, Campus Ring 6, D-28759 Bremen, Germany.
Iolyeva
Maria E
ME
Meyer-Grahle
Ulf
U
Brix
Klaudia
K
D000818:Animals; D016466:CHO Cells; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007422:Intestines / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics; D021381:Protein Transport
D018398:Homeodomain Proteins; D008562:Membrane Glycoproteins; D002401:Cathepsin B