In vitro non-viral murine pro-neurotrophin 3 gene transfer into rat bone marrow stromal cells.
Neurotrophin 3 (NT-3) is an important factor for promoting prenatal neural development, as well as regeneration, axogenesis and plasticity in postnatal life. Therapy with NT-3 was reported to improve the condition of patients suffering from degenerative diseases and traumatic injuries, however, the disadvantage of NT-3 protein delivery is its short half-life, thus our alternative approach is the use of NT-3 gene therapy. In this study, the bone marrow stromal cells (BMSCs) were isolated from adult rats, cultured for 4 passages and transfected with either pEGFP-N1 or a constructed vector containing murine proNT-3 (pSecTag2/HygroB-murine proNT-3) using Lipofectamine 2000 followed by Hygromycin B (200mg/kg). The transfection efficiency of the transiently transfected BMSCs was evaluated using the green fluorescence protein containing vector (pEGFP-N1). A quantitative evaluation of the NT-3 expression of mRNA using real time qRT-PCR shows that there was double fold increase in NT-3 gene expression compared with non-transfected BMSCs, also, the culture supernatant yielded double fold increase in NT-3 using ELISA technique, the data were supported by immunoblotting technique. This suggests that the use of this transfection technique can be useful for gene therapy in different neurological disorders with neurodegenerative or traumatic origins.
Cellular and Molecular Research Center, Qazvin University of Medical Science, Qazvin, Iran.
Shahram
S
Darabi
Department of Anatomical Sciences, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address: takialtr@modares.ac.ir.
Taki
T
Tiraihi
Department of Anatomy, Shahed University, Tehran, Iran.
AliReza
A
Delshad
Department of Genetics, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, Iran.
Majid
M
Sadeghizadeh
Department of Anatomical Sciences, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Wisam
W
Khalil
Shefa Neurosciences Research Center, Khatam Al-Anbia Hospital, Tehran, Iran.
Taher
T
Taheri
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001854:Bone Marrow Cells / Q000378:metabolism*; D002478:Cells, Cultured; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D020933:Neurotrophin 3 / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014162:Transfection
D015703:Antigens, CD; D020933:Neurotrophin 3; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins
BMSCs; NT-3; Non-viral transfection; Secretory vector; Transfection