J. Anim Sci. 2007. 85:E18-E19. doi:10.2527/jas.2006-486
© 2007 American Society of Animal Science
TRIENNIAL REPRODUCTION SYMPOSIUM |
Ribonucleic acid interference: A new approach to the in vivo study of gene function1
R. V. Anthony2 and
J. D. Cantlon
Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins 80523-1683
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Abstract
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The 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, along 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 and followed by a short poly(T) string to terminate transcription, are inserted downstream of an RNA polymerase III promoter within the viral vector of choice. Several viral vectors are useful for delivery of shRNA expression cassettes, each with particular attributes. 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, 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: ribonucleic acid interference • gene ablation • virus-mediated infection
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INTRODUCTION
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Characterization 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. Receptor agonists or antagonists can be used to study specific functions when available, but the time and expense of generating specific reagents without sufficient insight into a given molecule’s function is often prohibitive. Alternatively, during the past 15 to 20 yr, homologous recombination within mouse embryonic stem cell lines, providing for functional gene ablation or insertion, has been used extensively to define many gene functions in mice. Unfortunately, the use of similar methodologies in livestock species is not yet efficient, and the results obtained from rodents are not always applicable to livestock or humans. Consequently, there are still a fair number of gene products for which a clear definition of their function in livestock is lacking and, through the various animal genome projects, the number of genes without a known function in livestock is growing. A more recent technology that may well lend itself to in vivo gene function studies in livestock is the use of viral-mediated RNA interference. It is our objective to provide a brief overview of this technology.
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RNA INTERFERENCE
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Several approaches have been developed to target specific mRNA, with the aim of inhibiting translation through steric blocking or mRNA degradation. Introduction of DNA or RNA antisense oligonucleotides was reported over 20 yr ago (Stephenson and Zamecnik, 1978
; Mellon, 1985). The use of chemically modified oligonucleotides (e.g., morpholino-modified oligonucleotides) that block translation has improved the efficacy of this approach, and their use in livestock was reported recently (Dunlap et al., 2006). However, the parallel development of our understanding of naturally occurring RNA interference via microRNA (He and Hannon, 2004), along with the development of efficient viral-based vectors for gene transfer, may hold the greatest potential for specific gene loss-of-function studies in livestock.
When transcribed by cells, short-hairpin RNA (shRNA; Paddison et al., 2002) are recognized and processed in the same fashion as endogenous microRNA (He and Hannon, 2004). Cassettes encoding a shRNA, typically consisting of inverted repeats separated by a loop sequence and followed by a short poly(T) string to terminate transcription, are inserted downstream of an RNA polymerase III promoter within the viral vector of choice (Paddison et al. 2004). Several viral vectors are useful for delivery of shRNA expression cassettes, each with particular attributes. 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 21-base pair, double-stranded guide sequences. Guide sequences are incorporated into the RNA-induced silencing complex. Within the RNA-induced silencing complex, Argonaut 2 or Slicer cleaves the target RNA, which is then degraded. An animation of this process can be accessed at: http://www.nature.com/focus/rnai/animations/index.html (last accessed Oct. 5, 2006).
For each mRNA of interest, design and testing of 3 to 5 shRNA, along with adequate controls, usually are required to identify the most efficient construct before proceeding to in vivo use. Controls should include infection with virus derived from the empty expression vector, virus expressing a scrambled shRNA cassette, and if possible, virus encoding a shRNA cassette that has been shown to knock down expression of a different gene that does not affect the cell or tissue of interest. The reason behind these extensive controls is that there are a number of potential pitfalls that may negatively affect interpretation of the data. These include the failure to knock down expression of the specific mRNA of interest, generalized knockdown of the expression of many mRNA, or off-target effects. Furthermore, some double-stranded RNA may trigger interferon induction and the innate immune system, thereby compromising the results.
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PROSPECTIVE
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Whereas viral-mediated gene expression knockdown has been used effectively in rodents (Rubinson et al., 2003), its use in livestock has only been recently reported (Golding et al., 2006). In these studies, the prion protein concentration in the brain of a goat fetus generated by nuclear transfer was reduced by 90%. Furthermore, these authors (Golding et al., 2006) injected recombinant virus into the perivitelline space of cattle ova, and 76% of the resultant blastocysts expressed green fluorescent protein, a marker of viral-construct expression. The report by Golding et al. (2006) supports the idea that this technology may become an effective method for studying in vivo gene function in livestock.
As an alternative to injecting the shRNA-encoding virus into the perivitelline space, which could affect the developing fetus and the placenta, hatched blastocysts could be exposed to the virus, thereby directing infection to the trophectoderm (i.e., the placenta). Additionally, it may well be feasible to deliver the shRNA vector to specific organs or tissues in which the gene of interest is expressed (e.g., injection of follicles). Although the methodology is still in its experimental infancy, we believe that viral-mediated RNA interference has considerable potential for providing an efficient approach for the study of gene function in vivo in livestock.
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Footnotes
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1 Presented at the ADSA-ASAS Joint Annual Meeting, Triennial Reproduction Symposium: Molecular Techniques and Statistics, Minneapolis, MN, July 2006. Supported in part by National Research Initiative Competitive Grant No. 2005-35203-15885 from the USDA Cooperative State Research, Education, and Extension Service. 
2 Corresponding author: russ.anthony{at}colostate.edu
Received for publication July 20, 2006.
Accepted for publication September 22, 2006.
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LITERATURE CITED
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Dunlap, K. A., M. Palmarini, M. Varela, R. C. Burghardt, K. Hayashi, J. L. Farmer, and T. E. Spencer. 2006. Endogenous retroviruses regulate periimplantation placental growth and differentiation. Proc. Natl. Acad. Sci. USA 103:14390–14395.[Abstract/Free Full Text]
Golding, M. C., C. R. Long, M. A. Carmell, G. J. Hannon, and M. E. Westhusin. 2006. Suppression of prion protein in livestock by RNA interference. Proc. Natl. Acad. Sci. USA 103:5285–5290.[Abstract/Free Full Text]
He, L., and G. J. Hannon. 2004. MicroRNAs: Small RNAs with a big role in gene regulation. Nat. Rev. Genet. 5:522–531.[CrossRef][Medline]
Mellon, D. A. 1985. Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc. Natl. Acad. Sci. USA 82:144–148.[Abstract/Free Full Text]
Paddison, P. J., A. A. Caudy, E. Bernstein, G. J. Hannon, and D. S. Conklin. 2002. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16:948–958.[Abstract/Free Full Text]
Paddison, P. J., A. A. Caudy, R. Sachidanandam, and G. J. Hannon. 2004. Short hairpin activated gene silencing in mammalian cells. Methods Mol. Biol. 265:85–100.[Medline]
Rubinson, D. A., C. P. Dillon, A. V. Kwiatkowski, C. Sievers, L. Yang, J. Kopinja, M. Zhang, M. T. McManus, F. B. Gertler, M. L. Scott, and L. Van Parijs. 2003. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33:401–406.[CrossRef][Medline]
Stephenson, M. L., and P. C. Zamecnik. 1978. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. USA 75:285–288.[Abstract/Free Full Text]
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Abstract
INTRODUCTION
