Intestine-specific expression of green fluorescent protein-tagged cathepsin B: proof-of-principle experiments

2008 ◽  
Vol 0 (ja) ◽  
pp. 080808064931026-30
Author(s):  
Kristina Mayer ◽  
Maria E. Iolyeva ◽  
Ulf Meyer-Grahle ◽  
Klaudia Brix
2008 ◽  
Vol 389 (8) ◽  
Author(s):  
Kristina Mayer ◽  
Maria E. Iolyeva ◽  
Ulf Meyer-Grahle ◽  
Klaudia Brix

Abstract 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.


2001 ◽  
Vol 115 (6) ◽  
pp. 455-464 ◽  
Author(s):  
Xulun Zhang ◽  
Stephan L. Baader ◽  
Feng Bian ◽  
Wolfgang Müller ◽  
John Oberdick

2014 ◽  
Vol 60 (3) ◽  
pp. 230-237 ◽  
Author(s):  
Hitomi MATSUNARI ◽  
Toshihiro KOBAYASHI ◽  
Masahito WATANABE ◽  
Kazuhiro UMEYAMA ◽  
Kazuaki NAKANO ◽  
...  

1999 ◽  
Vol 41 (6) ◽  
pp. 675-684 ◽  
Author(s):  
Tomomi Yoshimizu ◽  
Noriyuki Sugiyama ◽  
Mario De Felice ◽  
Young Ii Yeom ◽  
Kazuyuki Ohbo ◽  
...  

FEBS Letters ◽  
1998 ◽  
Vol 440 (3) ◽  
pp. 370-376 ◽  
Author(s):  
M Fleischmann ◽  
W Bloch ◽  
E Kolossov ◽  
C Andressen ◽  
M Müller ◽  
...  

Endocrinology ◽  
2007 ◽  
Vol 148 (4) ◽  
pp. 1518-1523 ◽  
Author(s):  
Eisuke Itakura ◽  
Kousuke Odaira ◽  
Kotaro Yokoyama ◽  
Marumi Osuna ◽  
Takahiko Hara ◽  
...  

Folliculo-stellate (FS) cells are known to act as sustentacular cells or scavenger cells in the anterior lobe. However, the precise function and origin of FS cells are still under discussion. Like brain astrocytes, FS cells contain S-100β protein, and FS cells can be detected immunocytochemically using antibodies for S-100β protein after fixation; however, living FS cells can not be detected. The generation of transgenic rats expressing green fluorescent protein (GFP) under the control of S-100β protein gene promoter may allow the detection of living FS cells, which may be an excellent tool for the study of FS cells. With the aim of generation of transgenic rats, we analyzed the promoter activity of the S-100β gene and found that intron 1 is important for cell-specific expression of the S-100β gene. Therefore, we obtained a DNA construct containing GFP gene under a part of the S-100 promoter with intron 1. We transfected the construct into rat embryos and succeeded in generating transgenic rats. The transgenic rats expressed GFP in FS cells specifically in the anterior lobe. GFP is also expressed in other known S-100β-expressing cells, i.e. brain astrocytes, adipocytes, and chondrocytes. We believe that the newly generated transgenic rats will provide a new approach for the study of FS cells and other S-100β protein-producing cells.


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