Protocol

Knockdown Transgenic Mice Generated by Silencing Lentiviral Vectors: Zona Pellucida Removal and Subzonal Injection Methods

This protocol was adapted from "Knockdown Transgenic Mice Generated by Silencing Lentiviral Vectors," Chapter 75, 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

In order to generate transgenic and knockout animals, preimplantation embryos must be harvested and manipulated in vitro. Although the production of transgenic animals has been performed by pronuclear injection of DNA into single-cell embryos, the generation of mouse knockouts is time-consuming and laborious. An embryonic stem (ES) knockout line must be generated, characterized, and injected into a blastocyst in order to obtain a chimeric founder that can be subsequently bred to homozygosity. Taking advantage of the unique ability of lentiviral vectors to generate transgenic animals, one can use lentiviruses expressing short hairpin RNAs (shRNAs) from polymerase III (pol III) promoters such as H1 and mU6 to produce transgenic knockdown mice. This protocol describes two methods to deliver genes and small interfering RNA (siRNA)-expressing cassettes into preimplantation mouse embryos using lentiviral vectors: zona pellucida removal and subzonal injection. The zona pellucida removal method is able to achieve up to 100% transgenesis and does not require the use of a micromanipulator. However, zona removal is toxic to the embryos and results in low survival of embryos. The subzonal injection method is able to achieve up to 100% transgenesis and is not toxic for the embryos; therefore, survival of embryos is much higher. Nevertheless, it involves the use of a micromanipulator, which requires some skill.