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Knockdown Transgenic Protocol

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Selected Protocol from "Gene Transfer: Delivery and Expression of DNA and RNA", Edited by Theodore Friedmann and John Rossi.
Knockdown Transgenic Mice Generated by Silencing Lentiviral Vectors
Oded Singer, Gustavo Tiscornia, and Inder M. Verma
The Salk Institute for Biological Studies, Laboratory of Genetics, La Jolla, California 92037

This protocol describes the use of lentiviral vectors to deliver genes and small interfering RNA (siRNA)-expressing cassettes into preimplantation embryos. This technique allows for the rapid development of transgenic animals and transgenic knockdown animals.
For the purpose of generating transgenic and knockout animals, preimplantation embryos must be harvested and manipulated in vitro. Although the generation 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 (Lois et al. 2002; Pfeifer et al. 2002), lentiviruses expressing short hairpin RNAs (shRNAs) from polymerase III (pol III) promoters such as H1 (Tiscornia et al. 2003) and mU6 (Rubinson et al. 2003) can be used to generate transgenic knockdown mice. Two methods are available for delivering lentiviral particles to the embryo to obtain lentiviral transgenesis: zona pellucida removal and subzonal injection. For a detailed description of harvesting and manipulating mouse embryos, see Hogan et al. (1994). The zona pellucida removal protocol 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 protocol is able to achieve up to 100% transgenesis and is not toxic for the embryos; therefore, survival of embryos is much higher. Nevertheless, the use of micromanipulators requires some practice.

Pellucida Removal and Subzonal Injection Methods
This protocol describes two methods to deliver genes and siRNA-expressing cassettes into preimplantation mouse embryos using lentiviral vectors.

Products Available for this Protocol
Protocol Material Description Product #  Product Name Add to Cart
Buffers, Solutions, and Reagents      
H2O W4502 Water
NaCl S3014 Sodium chloride
KCl P9541 Potassium chloride
CaCl2•2H2O C7902 Calcium chloride dihydrate
MgCl2•6H2O M1028 Magnesium chloride solution
Glucose G5400 D-(+)-Glucose
Polyvinylpyrrolidone (PVP) P5288 Polyvinylpyrrolidone
Gonadotropin from pregnant mare serum G4527 Gonadotropin from pregnant mare serum
Human chorionic gonadotropin C8554 Chorionic gonadotropin human
Hyaluronidase solution H3884 Hyaluronidase from bovine testes
Mineral oil M5904 Mineral oil
M2 M7167 M2 medium
M16 M7292 M16 medium

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