Expression Liver-directed Genes by Employing Synthetic Transcriptional Control Units

Overview

Journal World J Gastroenterol

Publisher Baishideng Publishing Group

Specialty Gastroenterology

Date 2005 Sep 9

PMID 16149135

Citations 5

Authors

Marie-Luise Lemken

Wolfgang-A Wybranietz

Ulrike Schmidt

Florian Graepler

Sorin Armeanu

Michael Bitzer

Ulrich-M Lauer

Affiliations

Soon will be listed here.

Abstract

Aim: To generate and characterize the synthetic transcriptional control units for transcriptional targeting of the liver, thereby compensating for the lack of specificity of currently available gene therapeutic vector systems.

Methods: Synthetic transcriptional control unit constructs were generated and analyzed for transcriptional activities in different cell types by FACS quantification, semi-quantitative RT-PCR, and Western blotting.

Results: A new bifunctionally-enhanced green fluorescent protein (EGFP)/neo(r) fusion gene cassette was generated, and could flexibly be used both for transcript quantification and for selection of stable cell clones. Then, numerous synthetic transcriptional control units consisting of a minimal promoter linked to "naturally" derived composite enhancer elements from liver-specific expressed genes or binding sites of liver-specific transcription factors were inserted upstream of this reporter cassette. Following liposome-mediated transfection, EGFP reporter protein quantification by FACS analysis identified constructs encoding multimerized composite elements of the apolipoprotein B100 (ApoB) promoter or the ornithin transcarbamoylase (OTC) enhancer to exhibit maximum transcriptional activities in liver originating cell lines, but only background levels in non-liver originating cell lines. In contrast, constructs encoding only singular binding sites of liver-specific transcription factors, namely hepatocyte nuclear factor (HNF)1, HNF3, HNF4, HNF5, or CAAT/enhancer binding protein (C/EBP) only achieved background levels of EGFP expression. Finally, both semi-quantitative RT-PCR and Western blotting analysis of Hep3B cells demonstrated maximum transcriptional activities for a multimeric 4xApoB cassette construct, which fully complied with the data obtained by initial FACS analysis.

Conclusion: Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver-directed expression of therapeutic genes.

Citing Articles

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Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors.

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Optimized human factor IX expression cassettes for hepatic-directed gene therapy of hemophilia B.

Zhang R, Wang Q, Zhang L, Chen S Front Med. 2015; 9(1):90-9.

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Optimizing vector application for gene transfer into human hepatoblastoma cells.

Warmann S, Armeanu S, Heitmann H, Ruck P, Seitz G, Wessels J Pediatr Surg Int. 2006; 22(9):733-42.

PMID: 16896819 DOI: 10.1007/s00383-006-1727-3.

In vitro gene targeting in human hepatoblastoma.

Warmann S, Armeanu S, Frank H, Buck H, Graepler F, Lemken M Pediatr Surg Int. 2005; 22(1):16-23.

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