a sleeping beauty dna transposon-based genetic sensor for functional screening of vitamin d3 analogues睡美人dna transposon-based基因传感器功能筛查维生素d3的类似物.pdfVIP

Staunstrup et al. BMC Biotechnology 2011, 11:33 /1472-6750/11/33 RESEARCH ARTICLE Open Access A Sleeping Beauty DNA transposon-based genetic sensor for functional screening of vitamin D3 analogues 1 1 1 2 2 3 Nicklas H Staunstrup , Nynne Sharma , Rasmus O Bak , Lars Svensson , Thomas K Petersen , Lene Aarenstrup , Karsten Kristiansen4, Lars Bolund1 and Jacob Giehm Mikkelsen1* Abstract Background: Analogues 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. Results: The 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 insertio