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Rivista di Biologia Molecolare e Biotecnologie
Indexed/Abstracted in: EMBASE, Science Citation Index Expanded (SciSearch), Scopus
Impact Factor 0,246
Minerva Biotecnologica 2013 Giugno;25(2):75-80
The human tissue-plasminogen activator gene was transformed to cucumber cells
Asgari M. 1, Jalalijavaran M. 1, Moieni A. 1, Masoumiasl A. 2 ✉
1 Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran;
2 Department of biotechnology, Faculty of Agriculture, Yasuj University, Yasuj, Iran
Aim: Molecular farming has unlimited potential for the production of vaccines, antibodies and therapeutic proteins. Tissue plasminogen activator (tPA), is a serine protease that plays an important role in the fibrinolytic system and the dissolution of fibrin clots in humans. Because of the important and inevitable use of this medicine in cardiac diseases and its high price, the necessity of producing it in high quantities by genetic engineering is distinct.
Methods: In this study, the six hormonal treatments to verify the proper regeneration in cucumber (Esfahan variety) were used. Sensitivity of Cotyledon explants to kanamycin antibiotic was tested by culturing them in a selective medium with different concentrations of kanamycin. Then, the human tPA gene was cloned under the CaMV 35S promoter in pBI121 plasmid and was transferred to cucumber explants by Agrobacterium LBA4404. Regenerated plants on a selective medium were analyzed in both DNA and RNA levels.
Results and conclusion: The highest number of cotyledon explants regenerations were obtained in an MS medium with NAA (0.1 mgl-1) and BAP (3 mgl-1) hormones. The best concentration of kanamycin antibiotic to remove the non-transgenic cucumber plant cells was 40 mgl-1. Presence of the tPA gene in regenerated plants was confirmed by PCR reaction with specific primers. The tPA gene expression in transgenic plants was confirmed by RT-PCR analysis.