Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Summary

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.

Abstract

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.

Protocol

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

1. Make sure that there are plenty of cells available for transfection (several million cells for each DNA to be transfected).
2. Coat a tissue culture dish, into which the transfected cells will be se....

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
The Nucleofector Device	Tool	Amaxa	AAD-1001
Mouse neural stem cell nucleofector kit	Reagent	Amaxa	VPG-1004