Protocol

Genetic Manipulation of Mammalian Cells by Microinjection

This protocol was adapted from "Genetic Manipulation of Mammalian Cells by Microinjection," Chapter 71, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007.

INTRODUCTION

Microinjection allows the introduction of molecules into a defined population of cells at a defined concentration, and the timing of the experiment can be controlled stringently, minimizing problems associated with overexpression. Delivery by microinjection can be used for any type of cell that is adherent in culture, including primary cells. Perhaps its most powerful aspect is the ability to introduce several types of reagents into cells simultaneously, including DNA constructs, a labeled dextran to mark injected cells, antibodies, short interfering RNAs (siRNAs), and peptides. Because siRNA can be generated easily and rapidly for any target gene, it is relatively simple to assess many effects of the knockout of any gene in any type of adherent cell in a matter of days. However, microinjection is not trivial to learn, and requires attention to detail. Also, the limited number of cells involved often does not permit the subsequent analysis of effects on some biochemical parameters. While the major limitation of the approach is the small amount of material obtained, as the sensitivity of analysis techniques increases, so does the usefulness of this method. We emphasize here recent advances in the use of microinjection in combination with siRNA technology.