Construction Of Retroviral Vector Containing IGF-1 Gene And Its Expression In Rat Neural Stem Cells

Objective Neural stem cells (NSCs) are a subtype of tissue-specific progenitor cells that is capable of extended self-renewal and the ability to generate all major cell types of nervous tissue, such as neurons, astroglia and oligodendroglia. Targeted delivery and expression of growth factor genes such as IGF-1 in NSCs to construct genetic engineering NSCs is an important direction in gene therapy of nervous system disease. Insulin like growth factor-1 (IGF-1) is a polypeptide, it serves many critical functions in normal growth and development process of nervous system. Various kinds of trauma animal model including in vivo and in vitro have demonstrated its significant neurotrophy and neuroprotective effects. It's multieffect include maintenance of neuron growth, inhibition of apoptosis, promotion of cellular differentiation and regulation of blood-brain barrier permeability, neuronal excitability and participation of angiogenesis, etc. This experiment plans to construct a retroviral vector containing IGF-1 gene, then transfer it into NSCs, and detect the expression of IGF-1 gene. By means of that, to provide some experimental evidence for transgenic neural stem cell transplantation therapy of nervous system disease.Method 1. According to compatible restriction sites in pcDNA3.1-IGF-1 and pLXSN, we choose EcoR I and Xho I to digest the plasmid pcDNA3.1-IGF-1 and pLXSN. then use T4DNA ligation enzyme to connect cDNA and plasmid, Transform ligation mixture into E. coli JM109, incubate and pick separate colony, after propagation we extracte plasmids to perform restriction analysis to identify the construction.2. To acquire recombinant retrovirus, Lipofectamine was used to mediate recombinant plasmid pLXSN-IGF-1 transfection of PA317 packaging cells, Collect virus-containing medium from packaging cells, Infect NIH3T3 cells to determine viral titer. NSCs were...