Technology Review Cellscience Reviews Vol.3 No.1 ISSN 1742-8130

Reverse Genetics in Mammals Using RNAi

Reverse genetics in mammals has primarily relied upon targeted gene mutations by homologous recombination. Although highly effective, its application in wider biological studies has been hampered by drawbacks such as its high complexity, time-consuming nature, high cost and current limitation to mouse models. Recently, transgenic RNAi has been demonstrated to silence genes and to elicit phenotypes of gene dysfunction in vivo, indicating that it may provide an alternative method for reverse genetics in mice and other mammalian species.

Introduction

Reverse genetics studies gene function by targeted mutations and observation of the functional consequences. It is an indispensable vehicle in building a wealth of new knowledge in modern biology. Increasingly, it has been used to understand the mechanism of disease and to generate animal models for its study. Thus, advancements in the technology of reverse genetics have directly impacted upon human health. Since the early 1980s, reverse genetics in mammals have primarily relied upon gene knockouts in mice using homologous recombination technology. Although this method is highly effective and has been instrumental in defining gene function, several drawbacks have limited its use. First, this technology has only limited access through a few laboratories because of its technical complexity, high cost, and lengthy procedure. Even under ideal conditions, the process of obtaining a gene knockout mouse will take some 9-10 months (Beglopoulos & Shen, 2004). Due to problems inherent within experimentation, the time required to produce a gene knockout is often far longer. Second, such gene knockout technology is limited in its application to mice, and therefore cannot meet the demand for reverse genetics in other mammalian species. Third, gene knockout is not an ideal approach with which to generate disease models that require genetic hypomorphism, because standard gene knockouts often result in embryonic lethality in the homozygote and the absence of a phenotype in the heterozygote. To generate hypomorphic models, one has to knock in a mutant allele that has a reduced function. This approach has the same drawbacks of a standard gene knockout, and in addition requires prior knowledge of the mutant.

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