Abstract
RNA interference (RNAi) is a powerful gene knockdown technology that has been applied for functional genetic loss-of-function studies in many model eukaryotic systems, including embryonic stem cells (ESCs). Application of RNAi in ESCs allows for dissection of mechanisms by which ESCs self-renew and maintain pluripotency and also for specifying particular cell types needed for cell replacement therapies. Potent RNAi response can be induced by expression of a microRNA-embedded short-hairpin RNA (shRNAmir) cassette that is integrated in the genome by virus infection or site-specific recombination at a defined locus. In this chapter, I will provide detailed protocols to perform shRNAmir-mediated RNAi studies in mouse ESCs using retrovirus infection and loxP site-directed recombination for efficient constitutive and inducible gene knockdown, respectively.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156
Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 78:7634–7638
Chambers I, Smith A (2004) Self-renewal of teratocarcinoma and embryonic stem cells. Oncogene 23:7150–7160
Spagnoli FM, Hemmati-Brivanlou A (2006) Guiding embryonic stem cells towards differentiation: lessons from molecular embryology. Curr Opin Genet Dev 16:469–475
Odorico JS, Kaufman DS, Thomson JA (2001) Multilineage differentiation from human embryonic stem cell lines. Stem Cells 19:193–204
Gadue P, Huber TL, Nostro MC, Kattman S, Keller GM (2005) Germ layer induction from embryonic stem cells. Exp Hematol 33:955–964
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811
Ivanova N, Dobrin R, Lu R, Kotenko I, Levorse J, DeCoste C, Schafer X, Lun Y, Lemischka IR (2006) Dissecting self-renewal in stem cells with RNA interference. Nature 442:533–538
Ding L, Buchholz F (2006) RNAi in embryonic stem cells. Stem Cell Rev 2:11–18
Heidersbach A, Gaspar-Maia A, McManus MT, Ramalho-Santos M (2006) RNA interference in embryonic stem cells and the prospects for future therapies. Gene Ther 13:478–486
Spankuch B, Strebhardt K (2005) RNA interference-based gene silencing in mice: the development of a novel therapeutical strategy. Curr Pharm Des 11:3405–3419
Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448:313–317
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676
Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW, Daley GQ (2008) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451:141–146
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920
Chang K, Elledge SJ, Hannon GJ (2006) Lessons from nature: microRNA-based shRNA libraries. Nat Methods 3:707–714
Silva JM, Li MZ, Chang K, Ge W, Golding MC, Rickles RJ, Siolas D, Hu G, Paddison PJ, Schlabach MR, Sheth N, Bradshaw J, Burchard J, Kulkarni A, Cavet G, Sachidanandam R, McCombie WR, Cleary MA, Elledge SJ, Hannon GJ (2005) Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 37:1281–1288
Wang J, Rao S, Chu J, Shen X, Levasseur DN, Theunissen TW, Orkin SH (2006) A protein interaction network for pluripotency of embryonic stem cells. Nature 444:364–368
Wang J, Theunissen TW, Orkin SH (2007) Site-directed, virus-free, and inducible RNAi in embryonic stem cells. Proc Natl Acad Sci U S A 104:20850–20855
Lohmann F, Bieker JJ (2008) Activation of Eklf expression during hematopoiesis by Gata2 and Smad5 prior to erythroid commitment. Development 135:2071–2082
Conner DA (2001) Mouse embryo fibroblast (MEF) feeder cell preparation. Curr Protoc Mol Biol 23:22
Morita S, Kojima T, Kitamura T (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7:1063–1066
Gavrilescu LC, Van Etten RA (2007) Production of replication-defective retrovirus by transient transfection of 293T cells. J Vis Exp 10:550
Stegmeier F, Hu G, Rickles RJ, Hannon GJ, Elledge SJ (2005) A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci U S A 102:13212–13217
Kyba M, Perlingeiro RC, Daley GQ (2002) HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 109:29–37
Acknowledgments
The author would like to thank Dr. Stuart H. Orkin, an Investigator of Howard Hughes Medical Institute, for his support of the author’s postdoctoral training in his lab when the method was initially developed. The author’s current work is supported by the Seed Fund from the Black Family Stem Cell Institute in Mount Sinai School of Medicine.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Wang, J. (2017). Efficient Gene Knockdowns in Mouse Embryonic Stem Cells Using MicroRNA-Based shRNAs. In: Zhang, B. (eds) RNAi and Small Regulatory RNAs in Stem Cells. Methods in Molecular Biology, vol 1622. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7108-4_17
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7108-4_17
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7106-0
Online ISBN: 978-1-4939-7108-4
eBook Packages: Springer Protocols