Abstract
Random insertional mutagenesis and subsequent reverse genetic screening allow the isolation of mutants of interest. Here I describe the protocol for generating a tag insertion line and subsequent PCR-based screening for ARGONAUTE mutants as an example of a reverse genetic screen for the unicellular green alga Chlamydomonas reinhardtii.
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References
Meister G (2013) Argonaute proteins: functional insights and emerging roles. Nat Rev Genet 14(7):447–459. doi:10.1038/nrg3462
Casas-Mollano JA, Rohr J, Kim EJ, Balassa E, van Dijk K, Cerutti H (2008) Diversification of the core RNA interference machinery in Chlamydomonas reinhardtii and the role of DCL1 in transposon silencing. Genetics 179(1):69–81. doi:10.1534/genetics.107.086546
Cerutti H, Ma X, Msanne J, Repas T (2011) RNA-mediated silencing in algae: biological roles and tools for analysis of gene function. Eukaryot Cell 10(9):1164–1172. doi:10.1128/EC.05106-11
Kim EJ, Ma X, Cerutti H (2015) Gene silencing in microalgae: mechanisms and biological roles. Bioresour Technol 184:23–32. doi:10.1016/j.biortech.2014.10.119
Voshall A, Kim EJ, Ma X, Moriyama EN, Cerutti H (2015) Identification of AGO3-associated miRNAs and computational prediction of their targets in the green alga Chlamydomonas reinhardtii. Genetics 200(1):105–121. doi:10.1534/genetics.115.174797
Yamasaki T, Kim EJ, Cerutti H, Ohama T (2016) Argonaute3 is a key player in miRNA-mediated target cleavage and translational repression in Chlamydomonas. Plant J 85(2):258–268. doi:10.1111/tpj.13107
Gonzalez-Ballester D, Pootakham W, Mus F, Yang W, Catalanotti C, Magneschi L, de Montaigu A, Higuera JJ, Prior M, Galvan A, Fernandez E, Grossman AR (2011) Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants. Plant Methods 7:24. doi:10.1186/1746-4811-7-24
Berthold P, Schmitt R, Mages W (2002) An engineered Streptomyces hygroscopicus aph7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii Protist 153 (4):401–412. doi:10.1078/14344610260450136
Yamano T, Iguchi H, Fukuzawa H (2013) Rapid transformation of Chlamydomonas reinhardtii without cell-wall removal. J Biosci Bioeng 115(6):691–694. doi:10.1016/j.jbiosc.2012.12.020
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Yamasaki, T. (2017). Isolation and Characterization of ARGONAUTE Mutants in Chlamydomonas . In: Carbonell, A. (eds) Plant Argonaute Proteins. Methods in Molecular Biology, vol 1640. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7165-7_11
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DOI: https://doi.org/10.1007/978-1-4939-7165-7_11
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7164-0
Online ISBN: 978-1-4939-7165-7
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