Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications

Abstract

Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Optimum transfection efficiency and subsequent cell viability depend on a number of experimental variables such as cell density, liposome and DNA concentrations, liposome–DNA complexing time, and the presence or absence of media components such as antibiotics and serum. The importance of these factors in Lipofectamine 2000 mediated transfection will be discussed together with some specific applications: transfection of primary neurons, high throughput transfection, and delivery of small interfering RNAs.

Introduction

A very large number of techniques for the transfection of mammalian tissue culture cells have been described. The general requirements for a useful transfection reagent are that it is applied using simple, robust protocols to effect efficient transfection in a wide range of cell types and transfection formats, without excessive cytotoxicity. In this review, we discuss the requirements for successful transfection and the potential for optimization of transfection efficiency using Lipofectamine 2000.

For successful transfection a nucleic acid, which carries a net negative charge under normal physiological conditions, must come into contact with a cell membrane that also carries a net negative charge. Lipofectamine 2000 is a cationic liposome formulation that functions by complexing with nucleic acid molecules, allowing them to overcome the electrostatic repulsion of the cell membrane and to be taken up by the cell.

While there is a great deal of structural variation in lipid molecules that are able to mediate transfection, all have a number of common features [1]. First, there is a positively charged head group that usually contains one or more positively charged nitrogen atoms to allow interaction between the transfection reagent and the negatively charged sugar–phosphate backbone of a nucleic acid molecule. A spacer usually links the charged head group to one, two or three hydrocarbon chains. In some instances, this spacer may play a role in promoting contact between the cationic lipid and the nucleic acid. The hydrocarbon chains are often 14 or more carbon atoms in length. The degree of saturation and the presence of cis- or trans-forms introduce more potential for structural variations in these molecules.

The cationic lipid molecule is often formulated with a neutral co-lipid (helper lipid). Ideally a mixture of the two lipids is treated by a physical cavitation method such as sonication or microfluidization resulting in the formation of unilamellar liposomes of relatively uniform size (typically 100 nm in diameter). The positive charge on the surface of the liposome generates an electrostatic interaction with nucleic acids and facilitates contact with the negatively charged cell membrane. The neutral co-lipid mediates fusion of the liposome with the cell membrane effecting entry of the nucleic acid. To achieve expression of the transgene, DNA must reach the nucleus of the cell and become accessible to the transcriptional machinery. In actively dividing cells, transfected DNA may simply become trapped in the nucleus following the reassembly of the nuclear envelope at the end of mitosis [2], [3], [4]. However, Lipofectamine 2000 efficiently transfects post-mitotic neurons [5] and rat primary hepatocytes as well (S. Cates and B. Dalby, unpublished observations), suggesting that Lipofectamine 2000 may promote penetration of DNA through intact nuclear envelopes in these cell types, further demonstrating its utility.