title: Construction and quantitative evaluation of a dual specific promoter system for monitoring the expression status of Stra8 and c-kit genes.
abstract: Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.
authors.affiliation: Department of Biology, Faculty of Science, Ferdowsi University of Mashhad , Mashhad, Iran, Ma.Dastpak@stu.um.ac.ir.
authors.forename: Mahtab
authors.initials: M
authors.lastname: Dastpak
authors.forename: Maryam M
authors.initials: MM
authors.lastname: Matin
authors.forename: Moein
authors.initials: M
authors.lastname: Farshchian
authors.forename: Yvan
authors.initials: Y
authors.lastname: Arsenijevic
authors.forename: Madjid
authors.initials: M
authors.lastname: Momeni-Moghaddam
authors.forename: Sajjad
authors.initials: S
authors.lastname: Sisakhtnezhad
authors.forename: Sohrab
authors.initials: S
authors.lastname: Boozarpour
authors.forename: Hamid Reza
authors.initials: HR
authors.lastname: Bidkhori
authors.forename: Mahdi
authors.initials: M
authors.lastname: Mirahmadi
authors.forename: Ahmad Reza
authors.initials: AR
authors.lastname: Bahrami
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002460:Cell Line; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000502:physiology*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; D008164:Luminescent Proteins; C484447:STRA8 protein, human; D014212:Tretinoin; D019009:Proto-Oncogene Proteins c-kit; D004121:Dimethyl Sulfoxide