Abstract
To perform structure/function analyses of a protein in vivo, ideally one should be able to simultaneously abolish expression of the endogenous wild-type protein, substitute it with a form of the protein containing a targeted mutation, and analyze the functional consequences. Until recently, this was a highly challenging and/or laborious approach in mammalian systems, requiring a targeted gene knockin in a human cell line or mouse. Herein is described a RNA interference (RNAi)-based approach to achieve this much more simply in mammalian cells. A single retrovirus has been constructed, which directs expression of a short hairpin RNA (shRNA) to knockdown expression of the endogenous protein of interest; a cDNA coding for a wild-type or mutant version of the same protein that also contains “silent mutations” that do not affect the protein sequence, but do make the mRNA resistant to the shRNA; and a puromycin-resistance gene to allow rapid drug selection of the virus-infected cells. Using this virus, expression of the endogenous Anti-Silencing Function 1a (ASF1a) histone chaperone has been efficiently replaced in primary human cells, by an ectopically expressed epitope-tagged version. Moreover, the virus is designed so that other shRNA and shRNA-resistant cDNA cassettes can easily be substituted, making the approach readily applicable to other protein targets.
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References
Hannon, G. J. and Rossi, J. J. (2004) Unlocking the potential of the human genome with RNA interference. Nature 431, 371–378.
Paddison, P. J., Silva, J. M., Conklin, D. S., et al. (2004) A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431.
Westbrook, T. F., Martin, E. S., Schlabach, M. R., et al. (2005) A genetic screen for candidate tumor suppressors identifies REST. Cell 121, 837–848.
Berns, K., Hijmans, E. M., Mullenders, J., et al. (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437.
Nicke, B., Bastien, J., Khanna, S. J., et al. (2005) Involvement of MINK, a Ste20 family kinase, in Ras oncogene-induced growth arrest in human ovarian surface epithelial cells. Mol. Cell 20, 673–685.
Kolfschoten, I. G., van Leeuwen, B., Berns, K., et al. (2005) A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell 121, 849–858.
Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., and Conklin, D. S. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958.
Sambrook, J. and Russell, D. (Ed.) (2001) Molecular cloning: A laboratory manual. 3rd ed., Cold Spring Harbor Laboratory Press, NY.
Adams, P. D., Lopez, P., Sellers, W. R., and Kaelin, W. G., Jr. (1997) Fluorescence-activated cell sorting of transfected cells. Methods Enzymol. 283, 59–72.
Harlow, E. and Lane, D. (Ed.) (1988) Antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press, NY.
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Zhang, R., Adams, P.D., Ye, X. (2007). Design and Application of a shRNA-Based Gene Replacement Retrovirus. In: Ochs, M.F. (eds) Gene Function Analysis. Methods in Molecular Biology™, vol 408. Humana Press. https://doi.org/10.1007/978-1-59745-547-3_12
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DOI: https://doi.org/10.1007/978-1-59745-547-3_12
Publisher Name: Humana Press
Print ISBN: 978-1-58829-734-1
Online ISBN: 978-1-59745-547-3
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