Abstract
Genetic modification of mouse embryonic stem (ES) cells is a powerful technology that enabled the generation of a plethora of mutant mouse lines to study gene function and mammalian biology. Here we describe ES cell culture and transfection techniques used to manipulate the ES cell genome to obtain targeted ES cell clones. We include the standard gene targeting approach as well as the application of the CRISPR/Cas9 system that can improve the efficiency of homologous recombination in ES cells by introducing a double-strand DNA break at the target site.
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
References
Bradley A, Evans M, Kaufman MH, Robertson E (1984) Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309(5965):255–256
Capecchi MR (1989) Altering the genome by homologous recombination. Science 244(4910):1288–1292
Smih F, Rouet P, Romanienko PJ, Jasin M (1995) Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells. Nucleic Acids Res 23(24):5012–5019
Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG et al (2001) Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 21(1):289–297
Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP et al (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 29(8):731–734
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821
Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F et al (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910–918
Byrne SM, Ortiz L, Mali P, Aach J, Church GM (2015) Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Res 43(3):e21
Longo L, Bygrave A, Grosveld FG, Pandolfi PP (1997) The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism. Transgenic Res 6(5):321–328
Liu X, Wu H, Loring J, Hormuzdi S, Disteche CM, Bornstein P et al (1997) Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission. Dev Dyn 209(1):85–91
Codner GF, Lindner L, Caulder A, Wattenhofer-Donze M, Radage A, Mertz A et al (2016) Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction. BMC Cell Biol 17(1):30
Liang Q, Conte N, Skarnes WC, Bradley A (2008) Extensive genomic copy number variation in embryonic stem cells. Proc Natl Acad Sci U S A 105(45):17453–17456
Bryja V, Bonilla S, Cajanek L, Parish CL, Schwartz CM, Luo Y et al (2006) An efficient method for the derivation of mouse embryonic stem cells. Stem Cells 24(4):844–849
Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J et al (2008) The ground state of embryonic stem cell self-renewal. Nature 453(7194):519–523
Auerbach W, Dunmore JH, Fairchild-Huntress V, Fang Q, Auerbach AB, Huszar D et al (2000) Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines. Biotechniques 29(5):1024–8, 30, 32
Hansen GM, Markesich DC, Burnett MB, Zhu Q, Dionne KM, Richter LJ et al (2008) Large-scale gene trapping in C57BL/6N mouse embryonic stem cells. Genome Res 18(10):1670–1679
Seong E, Saunders TL, Stewart CL, Burmeister M (2004) To knockout in 129 or in C57BL/6: that is the question. Trends Genet 20(2):59–62
Ward CM, Barrow KM, Stern PL (2004) Significant variations in differentiation properties between independent mouse ES cell lines cultured under defined conditions. Exp Cell Res 293(2):229–238
Ware CB, Siverts LA, Nelson AM, Morton JF, Ladiges WC (2003) Utility of a C57BL/6 ES line versus 129 ES lines for targeted mutations in mice. Transgenic Res 12(6):743–746
Wong ES, Ban KH, Mutalif R, Jenkins NA, Copeland NG, Stewart CL (2010) A simple procedure for the efficient derivation of mouse ES cells. Methods Enzymol 476:265–283
Poueymirou WT, Auerbach W, Frendewey D, Hickey JF, Escaravage JM, Esau L et al (2007) F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses. Nat Biotechnol 25(1):91–99
Nagy A, Gocza E, Diaz EM, Prideaux VR, Ivanyi E, Markkula M et al (1990) Embryonic stem cells alone are able to support fetal development in the mouse. Development 110(3):815–821
Gertsenstein M, Nutter LM, Reid T, Pereira M, Stanford WL, Rossant J et al (2010) Efficient generation of germ line transmitting chimeras from C57BL/6N ES cells by aggregation with outbred host embryos. PLoS One 5(6):e11260
Behringer R, Gertsenstein M, Nagy K, Nagy A (2014) Manipulating the mouse embryo: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Kondoh G, Yamamoto Y, Yoshida K, Suzuki Y, Osuka S, Nakano Y et al (1999) Easy assessment of ES cell clone potency for chimeric development and germ-line competency by an optimized aggregation method. J Biochem Biophys Methods 39(3):137–142
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Gertsenstein, M., Mianné, J., Teboul, L., Nutter, L.M.J. (2020). Targeted Mutations in the Mouse via Embryonic Stem Cells. In: Larson, M. (eds) Transgenic Mouse. Methods in Molecular Biology, vol 2066. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9837-1_5
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9837-1_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9836-4
Online ISBN: 978-1-4939-9837-1
eBook Packages: Springer Protocols