A Sleeping Beauty DNA transposon-based genetic sensor for functional screening of vitamin D3 analogues

BMC Biotechnology

Research Article

Lene Aarenstrup¹  Karsten Kristiansen²  Lars Svensson³  Thomas K Petersen³  Jacob Giehm Mikkelsen⁴  Nynne Sharma⁴  Nicklas H Staunstrup⁴  Rasmus O Bak⁴  Lars Bolund⁴ 

[1] Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230, Odense M, Denmark;Department of Biology, University of Copenhagen, DK-2200, Copenhagen N, Denmark;Department of Disease Pharmacology, LEO Pharma, DK-2750, Ballerup, Denmark;Department of Human Genetics, University of Aarhus, DK-8000, Aarhus C, Denmark;

关键词: HaCaT Cell;    Alfacalcidol;    Calcipotriol;    Sleep Beauty;    HaCaT Keratinocyte Cell Line;   

DOI  :  10.1186/1472-6750-11-33

 received in 2010-12-20, accepted in 2011-04-07,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundAnalogues of vitamin D3 are extensively used in the treatment of various illnesses, such as osteoporosis, inflammatory skin diseases, and cancer. Functional testing of new vitamin D3 analogues and formulations for improved systemic and topical administration is supported by sensitive screening methods that allow a comparative evaluation of drug properties. As a new tool in functional screening of vitamin D3 analogues, we describe a genomically integratable sensor for sensitive drug detection. This system facilitates assessment of the pharmacokinetic and pharmadynamic properties of vitamin D3 analogues. The tri-cistronic genetic sensor encodes a drug-sensoring protein, a reporter protein expressed from an activated sensor-responsive promoter, and a resistance marker.ResultsThe three expression cassettes, inserted in a head-to-tail orientation in a Sleeping Beauty DNA transposon vector, are efficiently inserted as a single genetic entity into the genome of cells of interest in a reaction catalyzed by the hyperactive SB100X transposase. The applicability of the sensor for screening purposes is demonstrated by the functional comparison of potent synthetic analogues of vitamin D3 designed for the treatment of psoriasis and cancer. In clones of human keratinocytes carrying from a single to numerous insertions of the vitamin D3 sensor, a sensitive sensor read-out is detected upon exposure to even low concentrations of vitamin D3 analogues. In comparative studies, the sensor unveils superior potency of new candidate drugs in comparison with analogues that are currently in clinical use.ConclusionsOur findings demonstrate the use of the genetic sensor as a tool in first-line evaluation of new vitamin D3 analogues and pave the way for new types of drug delivery studies in sensor-transgenic animals.

【 授权许可】

Unknown   
© Staunstrup et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.