Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.
Note: Refer to the Passaging Human Neural Stem Cells article (https://www.jove.com/index/Details.stp?ID=263) and the Counting Human Neural Stem Cells article (https://www.jove.com/index/Details.stp?ID=262) to learn how to resuspend and count hNSPCs.
Preparation
Transfection
This protocol describes a relatively rapid and efficient transfection procedure for hNSPCs. Using this procedure, we have obtained initial transfection efficiencies of ~35%. Cells transfected by this procedure can be used for short-term studies (transiently transfected cells) or for longer-term studies if a selection agent is used to generate a stably transfected population. We have generated stably transfected cells in which ~70% of the cells express the exogenous protein. In transiently transfected hNSPCs, we have observed continued expression of the exogenous protein for ~7 days.
The authors would like to acknowledge Dr. Philip H. Schwartz of the National Human Neural Stem Cell Resource at the Children s Hospital, Orange County Research Institute for providing hNSPC cultures.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
The Nucleofector Device | Tool | Amaxa | AAD-1001 | |
Mouse neural stem cell nucleofector kit | Reagent | Amaxa | VPG-1004 |