Abstract
One of the remarkable achievements in knockout (KO) rat production reported during the period 2008–2010 is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing glycogen synthase kinase 3 and mitogen-activated protein kinase kinase inhibitors (2i medium). Here, we report gene-targeting technology via homologous recombination in rat ES cells, demonstrating its use through production of a protease-activated receptor-2 gene (Par-2) KO rat. We began by generating germline-competent ES cells from Dark Agouti rats using 2i medium. These ES cells, which differentiate into cardiomyocytes in vitro, can produce chimeras with high ES cell contribution when injected into blastocysts. We then introduced a targeting vector with a neomycin-resistant gene driven by the CAG promoter to disrupt Par-2. After a 7-day drug selection, 489 neomycin-resistant colonies were obtained. Following screening by polymerase chain reaction (PCR) genotyping and quantitative PCR analysis, we confirmed three homologous recombinant clones, resulting in chimeras that transmitted the Par-2 targeted allele to offspring. Par-2 KO rats showed a loss of Par-2 messenger RNA expression in their stomach cells and a lack of PAR-2 mediated smooth muscle relaxation in the aorta as indicated by pharmacological testing. Compared with mice, rats offer many advantages in biomedical research, including a larger body size; consequently, they are widely used in scientific investigation. Thus, the establishment of a gene-targeting technology using rat ES cells will be a valuable tool in human disease model production and drug discovery.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Buehr M, Meek S, Blair K, Yang J, Ure J, Silva J, McLay R, Hall J, Ying QL, Smith A (2008) Capture of authentic embryonic stem cells from rat blastocysts. Cell 135:1287–1298
Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6:507–512
Cocks TM, Fong B, Chow JM, Anderson GP, Frauman AG, Goldie RG, Henry PJ, Carr MJ, Hamilton JR, Moffatt JD (1999) A protective role for protease-activated receptors in the airways. Nature 398:156–160
Consortium International Mouse Knockout, Collins FS, Rossant J, Wurst W (2007) A mouse for all reasons. Cell 128:9–13
Consortium STAR, Saar K, Beck A, Bihoreau MT, Birney E, Brocklebank D, Chen Y, Cuppen E, Demonchy S, Dopazo J, Flicek P, Foglio M, Fujiyama A, Gut IG, Gauguier D, Guigo R, Guryev V, Heinig M, Hummel O, Jahn N, Klages S, Kren V, Kube M, Kuhl H, Kuramoto T, Kuroki Y, Lechner D, Lee YA, Lopez-Bigas N, Lathrop GM, Mashimo T, Medina I, Mott R, Patone G, Perrier-Cornet JA, Platzer M, Pravenec M, Reinhardt R, Sakaki Y, Schilhabel M, Schulz H, Serikawa T, Shikhagaie M, Tatsumoto S, Taudien S, Toyoda A, Voigt B, Zelenika D, Zimdahl H, Hubner N (2008) SNP and haplotype mapping for genetic analysis in the rat. Nat Genet 40:560–566
Cui X, Ji D, Fisher DA, Wu Y, Briner DM, Weinstein EJ (2011) Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat Biotechnol 29:64–67
Damiano BP, Cheung WM, Santulli RJ, Fung-Leung WP, Ngo K, Ye RD, Darrow AL, Derian CK, de Garavilla L, Andrade-Gordon P (1999) Cardiovascular responses mediated by protease-activated receptor-2 (PAR-2) and thrombin receptor (PAR-1) are distinguished in mice deficient in PAR-2 or PAR-1. J Pharmacol Exp Ther 288:671–678
Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156
Feero WG, Guttmacher AE, Collins FS (2010) Genomic medicine—an updated primer. N Engl J Med 362:2001–2011
Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, Vincent A, Lam S, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Ménoret S, Anegon I, Davis GD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R (2009) Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325:433
Hirabayashi M, Kato M, Kobayashi T, Sanbo M, Yagi T, Hochi S, Nakauchi H (2010) Establishment of rat embryonic stem cell lines that can participate in germline chimerae at high efficiency. Mol Reprod Dev 77:94
Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC, Katibah GE, Amora R, Boydston EA, Zeitler B, Meng X, Miller JC, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jaenisch R (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27:851–857
Izsvák Z, Fröhlich J, Grabundzija I, Shirley JR, Powell HM, Chapman KM, Ivics Z, Hamra FK (2010) Generating knockout rats by transposon mutagenesis in spermatogonial stem cells. Nat Methods 7:443–445
Jacob HJ, Lazar J, Dwinell MR, Moreno C, Geurts AM (2010) Gene targeting in the rat: advances and opportunities. Trends Genet 26:510–518
Katada H, Chen HJ, Shigi N, Komiyama M (2009) Homologous recombination in human cells using artificial restriction DNA cutter. Chem Commun (Camb) (43):6545–6547
Kawabata A (2003) Gastrointestinal functions of proteinase-activated receptors. Life Sci 74:247–254
Kawabata A, Kubo S, Nakaya Y, Ishiki T, Kuroda R, Sekiguchi F, Kawao N, Nishikawa H (2004) Distinct roles for protease-activated receptors 1 and 2 in vasomotor modulation in rat superior mesenteric artery. Cardiovasc Res 61:683–692
Kawagoe J, Takizawa T, Matsumoto J, Tamiya M, Meek SE, Smith AJ, Hunter GD, Plevin R, Saito N, Kanke T, Fujii M, Wada Y (2002) Effect of protease-activated receptor-2 deficiency on allergic dermatitis in the mouse ear. Jpn J Pharmacol 88:77–84
Kawamata M, Ochiya T (2010) Generation of genetically modified rats from embryonic stem cells. Proc Natl Acad Sci USA 107:14223–14228
Kitada K, Ishishita S, Tosaka K, Takahashi R, Ueda M, Keng VW, Horie K, Takeda J (2007) Transposon-tagged mutagenesis in the rat. Nat Methods 4:131–133
Li P, Tong C, Mehrian-Shai R, Jia L, Wu N, Yan Y, Maxson RE, Schulze EN, Song H, Hsieh CL, Pera MF, Ying QL (2008) Germline competent embryonic stem cells derived from rat blastocysts. Cell 135:1299–1310
Lindner JR, Kahn ML, Coughlin SR, Sambrano GR, Schauble E, Bernstein D, Foy D, Hafezi-Moghadam A, Ley K (2000) Delayed onset of inflammation in protease-activated receptor-2-deficient mice. J Immunol 165:6504–6510
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 USA 78:7634–7638
McNeish J (2004) Embryonic stem cells in drug discovery. Nat Rev Drug Discov 3:70–80
Nagy A, Gócza E, Diaz EM, Prideaux VR, Iványi E, Markkula M, Rossant J (1990) Embryonic stem cells alone are able to support fetal development in the mouse. Development. 110:815–821
Nichols J, Ying QL (2006) Derivation and propagation of embryonic stem cells in serum- and feeder-free culture. Methods Mol Biol 329:91–98
Shinozawa T, Tsuji A, Imahashi K, Nakashima K, Sawada H, Toyoshiba H, Yamamoto S, Takami K, Imai R (2009) Gene expression profiling of functional murine embryonic stem cell-derived cardiomyocytes and comparison with adult heart: profiling of murine ESC-derived cardiomyocytes. J Biomol Screen 14:239–245
Tong C, Li P, Wu NL, Yan Y, Ying QL (2010) Production of p53 gene knockout rats by homologous recombination in embryonic stem cells. Nature 467:211–213
Valenzuela DM, Murphy AJ, Frendewey D, Gale NW, Economides AN, Auerbach W, Poueymirou WT, Adams NC, Rojas J, Yasenchak J, Chernomorsky R, Boucher M, Elsasser AL, Esau L, Zheng J, Griffiths JA, Wang X, Su H, Xue Y, Dominguez MG, Noguera I, Torres R, Macdonald LE, Stewart AF, DeChiara TM, Yancopoulos GD (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21:652–659
Ying QL, Smith AG (2003) Defined conditions for neural commitment and differentiation. Methods Enzymol 365:327–341
Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A (2008) The ground state of embryonic stem cell self-renewal. Nature 453:519–523
Zambrowicz BP, Sands AT (2003) Knockouts model the 100 best-selling drugs—will they model the next 100? Nat Rev Drug Discov 2:38–51
Zan Y, Haag JD, Chen KS, Shepel LA, Wigington D, Wang YR, Hu R, Lopez-Guajardo CC, Brose HL, Porter KI, Leonard RA, Hitt AA, Schommer SL, Elegbede AF, Gould MN (2003) Production of knockout rats using ENU mutagenesis and a yeast-based screening assay. Nat Biotechnol 21:645–651
Zhou Q, Renard JP, Le Friec G, Brochard V, Beaujean N, Cherifi Y, Fraichard A, Cozzi J (2003) Generation of fertile cloned rats by regulating oocyte activation. Science 302:1179
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Yamamoto, S., Nakata, M., Sasada, R. et al. Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats. Transgenic Res 21, 743–755 (2012). https://doi.org/10.1007/s11248-011-9564-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-011-9564-0