Transfecting human neural stem cells with the Amaxa Nucleofector.
Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency. Other mechanical methods of introducing a gene of interest, such as a "gene gun" or microinjection, are also limited by poor cell viability and low numbers of transfected cells. The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns. We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells. Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection. The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.
Marchenko
Steven
S
Department of Pathology, University of California, Irvine, USA. smarchen@uci.edu
Flanagan
Lisa
L
D002021:Buffers; D004247:DNA / Q000378:metabolism*; D018274:Electroporation / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D002021:Buffers; D004247:DNA