title: Generation of transgenic mesenchymal stem cells expressing green fluorescent protein as reporter gene using no viral vector in caprine.
abstract: Mesenchymal stromal cells (MSC) are multipotent cells that can be derived from many different organs and tissues. While there are many ways to label and track cells each with strengths and weakness, the green fluorescent protein (GFP) is a reporter gene commonly employed. In the present study, caprine MSC were collected from bone marrow and cells were characterised with MSC specific markers. Passage 10 (P10) MSC cells were transfected using plasmid vector containing GFP as reporter gene with different concentrations of DNA and lipofectamine. Six different concentrations of DNA and lipofectamine as 1 microg DNA: 2 microL lipofectamine, 1 microg DNA: 2.5 microL lipofectamine, 1.2 microg DNA: 2.2 microL lipofectamine, 1.2 microg DNA: 2.5 microL lipofectamine, 1.5 microg DNA: 2.5 microL lipofectamine, 1.5 microg DNA: 3 microL lipofectamine were used. After 24 h and 48 h of transfection, caprine MSC were observed under florescent microscope. Highest transfection rate indicating green flourecscent MSC were found when the cells were transfected with 1.2 microg DNA: 2.2 microL lipofectamine and 1.5 microg DNA: 2.5 microL lipofectamine than other combinations. These cells have been propagated beyond 4th passage maintaining GFP expression. The results indicated that stable GFP positive MSC cells can be generated using the above protocol. These cells are being used for transplantation studies.
authors.affiliation: Division of Physiology & Climatology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India. manish.vet82@gmail.com
authors.forename: Manish
authors.initials: M
authors.lastname: Kumar
authors.forename: T
authors.initials: T
authors.lastname: Yasotha
authors.forename: R K
authors.initials: RK
authors.lastname: Singh
authors.forename: Renu
authors.initials: R
authors.lastname: Singh
authors.forename: Kuldeep
authors.initials: K
authors.lastname: Kumar
authors.forename: R
authors.initials: R
authors.lastname: Ranjan
authors.forename: Chetan D
authors.initials: CD
authors.lastname: Meshram
authors.forename: B C
authors.initials: BC
authors.lastname: Das
authors.forename: Sadhan
authors.initials: S
authors.lastname: Bag
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis*; D002478:Cells, Cultured; D005822:Genetic Vectors; D006041:Goats; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007621:Karyotyping; D008055:Lipids; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D015415:Biomarkers; D008055:Lipids; C086724:Lipofectamine; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins