RNA Interference: A New Approach to In Vivo Study of Gene Function

¹ Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1683

^* To whom correspondence should be addressed. E-mail: russ.anthony{at}colostate.edu.

	Abstract

Definition of hormone function was classically accomplished by ablation-replacement studies. However, as our knowledge of the complexity of hormones and growth factors has grown, it has become increasingly difficult to clearly define the necessity and function of many of the hormones, growth factors, and regulatory proteins under investigation. The use of homologous recombination within mouse embryonic stem cell lines allows functional gene ablation, and has been used extensively during the past 15 yr to define specific gene function. The use of similar methodologies in livestock species has yet to yield an efficient approach. In contrast, the parallel development of our understanding of naturally occurring RNA interference with the development of efficient virus-based vectors for gene transfer holds great potential for effectively 'knocking down' specific gene function. Short-hairpin (sh) RNA-encoding cassettes, typically consisting of inverted repeats separated by a loop sequence, followed by a short poly(T) string to terminate transcription, are inserted downstream of a RNA polymerase III promoter within the viral-vector of choice. Several virus vectors are useful for delivery of shRNA expression cassettes, each with particular attributes. Both adenovirus and lentivirus-derived vectors provide a high rate of infectivity in most mammalian cell types, with lentiviral vectors allowing stable integration into the host genome if the study of long-term effects is needed. Upon transcription a shRNA is generated and the loop is recognized by the processing enzyme Dicer, generating 'guide' sequences. Guide sequences are incorporated into the RNA-induced silencing complex (RISC), which targets mRNA for degradation if recognized by the guide sequence. For each mRNA of interest, design and testing of a number of shRNA, along with adequate controls, are required to identify the most efficient construct before proceeding to in vivo use. This technology may become the method of choice for defining gene function in livestock.

Key Words: RNA interference, short hairpin RNA, gene ablation, virus-mediated infection