Skip to main content
SpringerLink
Log in

Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in hematological parameters

  • Published:
Mammalian Genome Aims and scope Submit manuscript

Abstract

Research on hematological disorders relies on suitable animal models. We retrospectively evaluated the use of the hematological parameters hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT) in the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project as parameters for the generation of novel animal models for human diseases. The analysis was carried out on more than 16,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the levels of the chosen parameters. Identification of animals exhibiting altered values and transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters MCV, RBC, and PLT. Analysis of the causative mutation was started in selected lines, thereby revealing a novel mutation in the transferrin receptor gene (Tfrc) in one line. Thus, novel phenotype-driven mouse models were established to analyze the genetic components of hematological disorders.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  • Aigner B, Rathkolb B, Klaften M, Sedlmeier R, Klempt M, Wagner S, Michel D, Mayer U, Klopstock T, Hrabé de Angelis M, Wolf E (2009) Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in plasma enzyme activities as novel organ-specific disease models. Exp Physiol 94:412–421

    Article  PubMed  CAS  Google Scholar 

  • Alexander WS, Viney EM, Zhang JG, Metcalf D, Kauppi M, Hyland CD, Carpinelli MR, Stevenson W, Croker BA, Hilton AA, Ellis S, Selan C, Nandurkar HH, Goodnow CC, Kile BT, Nicola NA, Roberts AW, Hilton DJ (2006) Thrombocytopenia and kidney disease in mice with a mutation in the C1galt1 gene. Proc Natl Acad Sci USA 103:16442–16447

    Article  PubMed  CAS  Google Scholar 

  • Andrews NC (2009) Genes determining blood cell traits. Nat Genet 41:1161–1162

    Article  PubMed  CAS  Google Scholar 

  • Barbaric I, Wells S, Russ A, Dear TN (2007) Spectrum of ENU-induced mutations in phenotype-driven and gene-driven screens in the mouse. Environ Mol Mutagen 48:124–142

    Article  PubMed  CAS  Google Scholar 

  • Boles MK, Wilkinson BM, Wilming LG, Liu B, Probst FJ, Harrow J, Grafham D, Hentges KE, Woodward LP, Maxwell A, Mitchell K, Risley MD, Johnson R, Hirschi K, Lupski JR, Funato Y, Miki H, Marin-Garcia P, Matthews L, Coffey AJ, Parker A, Hubbard TJ, Rogers J, Bradley A, Adams DJ, Justice MJ (2009) Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin. PLoS Genet 5:e1000759

    Article  PubMed  Google Scholar 

  • Carpinelli MR, Hilton DJ, Metcalf D, Antonchuk JL, Hyland CD, Mifsud SL, Di Rago L, Hilton AA, Willson TA, Roberts AW, Ramsay RG, Nicola NA, Alexander WS (2004) Suppressor screen in Mpl-/- mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling. Proc Natl Acad Sci USA 101:6553–6558

    Article  PubMed  CAS  Google Scholar 

  • Champy MF, Selloum M, Zeitler V, Caradec C, Jung B, Rousseau S, Pouilly L, Sorg T, Auwerx J (2008) Genetic background determines metabolic phenotypes in the mouse. Mamm Genome 19:318–331

    Article  PubMed  CAS  Google Scholar 

  • Chan ER, Lavender H, Li G, Haviernik P, Bunting KD, Adams MD (2009) An ENU-induced recessive mutation in Mpl leads to thrombocytopenia with overdominance. Exp Hematol 37:276–284

    Article  PubMed  CAS  Google Scholar 

  • Cordes SP (2005) N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express. Microbiol Mol Biol Rev 69:426–439

    Article  PubMed  CAS  Google Scholar 

  • Du X, She E, Gelbart T, Truksa J, Lee P, Xia Y, Khovananth K, Mudd S, Mann N, Moresco EM, Beutler E, Beutler B (2008) The serine protease TMPRSS6 is required to sense iron deficiency. Science 320:1088–1092

    Article  PubMed  CAS  Google Scholar 

  • Feng J, Wang H, Morse HC III (2007) Functional deficiency in IL-7 caused by an N-ethyl-N-nitrosourea-induced point mutation. Genetics 175:545–551

    Article  PubMed  CAS  Google Scholar 

  • Gailus-Durner V, Fuchs H, Becker L, Bolle I, Brielmeier M, Calzada-Wack J, Elvert R, Ehrhardt N, Dalke C, Franz TJ, Grundner-Culemann E, Hammelbacher S, Holter SM, Holzlwimmer G, Horsch M, Javaheri A, Kalaydjiev SV, Klempt M, Kling E, Kunder S, Lengger C, Lisse T, Mijalski T, Naton B, Pedersen V, Prehn C, Przemeck G, Racz I, Reinhard C, Reitmeir P, Schneider I, Schrewe A, Steinkamp R, Zybill C, Adamski J, Beckers J, Behrendt H, Favor J, Graw J, Heldmaier G, Hofler H, Ivandic B, Katus H, Kirchhof P, Klingenspor M, Klopstock T, Lengeling A, Muller W, Ohl F, Ollert M, Quintanilla-Martinez L, Schmidt J, Schulz H, Wolf E, Wurst W, Zimmer A, Busch DH, Hrabé de Angelis M (2005) Introducing the German Mouse Clinic: open access platform for standardized phenotyping. Nat Methods 2:403–404

    Article  PubMed  CAS  Google Scholar 

  • Ganesh SK, Zakai NA, van Rooij FJ, Soranzo N, Smith AV, Nalls MA, Chen MH, Kottgen A, Glazer NL, Dehghan A, Kuhnel B, Aspelund T, Yang Q, Tanaka T, Jaffe A, Bis JC, Verwoert GC, Teumer A, Fox CS, Guralnik JM, Ehret GB, Rice K, Felix JF, Rendon A, Eiriksdottir G, Levy D, Patel KV, Boerwinkle E, Rotter JI, Hofman A, Sambrook JG, Hernandez DG, Zheng G, Bandinelli S, Singleton AB, Coresh J, Lumley T, Uitterlinden AG, Vangils JM, Launer LJ, Cupples LA, Oostra BA, Zwaginga JJ, Ouwehand WH, Thein SL, Meisinger C, Deloukas P, Nauck M, Spector TD, Gieger C, Gudnason V, van Duijn CM, Psaty BM, Ferrucci L, Chakravarti A, Greinacher A, O’Donnell CJ, Witteman JC, Furth S, Cushman M, Harris TB, Lin JP (2009) Multiple loci influence erythrocyte phenotypes in the Charge Consortium. Nat Genet 41:1191–1198

    Article  PubMed  CAS  Google Scholar 

  • Greig KT, Antonchuk J, Metcalf D, Morgan PO, Krebs DL, Zhang JG, Hacking DF, Bode L, Robb L, Kranz C, de Graaf C, Bahlo M, Nicola NA, Nutt SL, Freeze HH, Alexander WS, Hilton DJ, Kile BT (2007) Agm1/Pgm3-mediated sugar nucleotide synthesis is essential for hematopoiesis and development. Mol Cell Biol 27:5849–5859

    Article  PubMed  CAS  Google Scholar 

  • Grosse J, Braun A, Varga-Szabo D, Beyersdorf N, Schneider B, Zeitlmann L, Hanke P, Schropp P, Muhlstedt S, Zorn C, Huber M, Schmittwolf C, Jagla W, Yu P, Kerkau T, Schulze H, Nehls M, Nieswandt B (2007) An EF hand mutation in Stim1 causes premature platelet activation and bleeding in mice. J Clin Invest 117:3540–3550

    Article  PubMed  CAS  Google Scholar 

  • Hrabé de Angelis M, Flaswinkel H, Fuchs H, Rathkolb B, Soewarto D, Marschall S, Heffner S, Pargent W, Wuensch K, Jung M, Reis A, Richter T, Alessandrini F, Jakob T, Fuchs E, Kolb H, Kremmer E, Schaeble K, Rollinski B, Roscher A, Peters C, Meitinger T, Strom T, Steckler T, Holsboer F, Klopstock T, Gekeler F, Schindewolf C, Jung T, Avraham K, Behrendt H, Ring J, Zimmer A, Schughart K, Pfeffer K, Wolf E, Balling R (2000) Genome-wide, large-scale production of mutant mice by ENU mutagenesis. Nat Genet 25:444–447

    Article  PubMed  Google Scholar 

  • Hrabé de Angelis M, Michel D, Wagner S, Becker S, Beckers J (2007) Chemical mutagenesis in mice. In: Fox JG, Barthold SW, Davisson MT, Newcomer CE, Quimby FW, Smith AL (eds) The mouse in biomedical research. History, wild mice, and genetics, vol 1. Academic Press, Burlington, pp 225–260

    Google Scholar 

  • Huang HW, Chiang YJ, Hung SI, Li CL, Yen JJ (2007) An IL-7 splicing-defect lymphopenia mouse model revealed by genome-wide mutagenesis. J Biomed Sci 14:169–181

    Article  PubMed  CAS  Google Scholar 

  • Kauppi M, Murphy JM, de Graaf CA, Hyland CD, Greig KT, Metcalf D, Hilton AA, Nicola NA, Kile BT, Hilton DJ, Alexander WS (2008) Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl-/- mice. Blood 112:3148–3153

    Article  PubMed  CAS  Google Scholar 

  • Keays DA, Clark TG, Campbell TG, Broxholme J, Valdar W (2007) Estimating the number of coding mutations in genotypic and phenotypic driven N-ethyl-N-nitrosourea (ENU) screens: revisited. Mamm Genome 18:123–124

    Article  PubMed  Google Scholar 

  • Kile BT, Hentges KE, Clark AT, Nakamura H, Salinger AP, Liu B, Box N, Stockton DW, Johnson RL, Behringer RR, Bradley A, Justice MJ (2003a) Functional genetic analysis of mouse chromosome 11. Nature 425:81–86

    Article  PubMed  CAS  Google Scholar 

  • Kile BT, Mason-Garrison CL, Justice MJ (2003b) Sex and strain-related differences in the peripheral blood cell values of inbred mouse strains. Mamm Genome 14:81–85

    Article  PubMed  Google Scholar 

  • Kile BT, Panopoulos AD, Stirzaker RA, Hacking DF, Tahtamouni LH, Willson TA, Mielke LA, Henley KJ, Zhang JG, Wicks IP, Stevenson WS, Nurden P, Watowich SS, Justice MJ (2007) Mutations in the cofilin partner Aip1/Wdr1 cause autoinflammatory disease and macrothrombocytopenia. Blood 110:2371–2380

    Article  PubMed  CAS  Google Scholar 

  • Klempt M, Rathkolb B, Aigner B, Wolf E (2006a) Clinical chemical screen. In: Hrabé de Angelis M, Chambon P, Brown S (eds) Standards of mouse model phenotyping. Wiley-VCH, Weinheim, pp 87–107

    Chapter  Google Scholar 

  • Klempt M, Rathkolb B, Fuchs E, Hrabé de Angelis M, Wolf E, Aigner B (2006b) Genotype-specific environmental impact on the variance of blood values in inbred and F1 hybrid mice. Mamm Genome 17:93–102

    Article  PubMed  Google Scholar 

  • Lambe T, Simpson RJ, Dawson S, Bouriez-Jones T, Crockford TL, Lepherd M, Latunde-Dada GO, Robinson H, Raja KB, Campagna DR, Villarreal G Jr, Ellory JC, Goodnow CC, Fleming MD, McKie AT, Cornall RJ (2009) Identification of a Steap3 endosomal targeting motif essential for normal iron metabolism. Blood 113:1805–1808

    Article  PubMed  CAS  Google Scholar 

  • Levy JE, Jin O, Fujiwara Y, Kuo F, Andrews NC (1999) Transferrin receptor is necessary for development of erythrocytes and the nervous system. Nat Genet 21:396–399

    Article  PubMed  CAS  Google Scholar 

  • Loeb WF, Quimby FW (1999) The clinical chemistry of laboratory animals. Taylor and Francis, Philadelphia

    Google Scholar 

  • Loughran SJ, Kruse EA, Hacking DF, de Graaf CA, Hyland CD, Willson TA, Henley KJ, Ellis S, Voss AK, Metcalf D, Hilton DJ, Alexander WS, Kile BT (2008) The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells. Nat Immunol 9:810–819

    Article  PubMed  CAS  Google Scholar 

  • Majewski IJ, Metcalf D, Mielke LA, Krebs DL, Ellis S, Carpinelli MR, Mifsud S, Di Rago L, Corbin J, Nicola NA, Hilton DJ, Alexander WS (2006) A mutation in the translation initiation codon of Gata-1 disrupts megakaryocyte maturation and causes thrombocytopenia. Proc Natl Acad Sci USA 103:14146–14151

    Article  PubMed  CAS  Google Scholar 

  • Majewski IJ, Blewitt ME, de Graaf CA, McManus EJ, Bahlo M, Hilton AA, Hyland CD, Smyth GK, Corbin JE, Metcalf D, Alexander WS, Hilton DJ (2008) Polycomb repressive complex 2 (PRC2) restricts hematopoietic stem cell activity. PLoS Biol 6:e93

    Article  PubMed  Google Scholar 

  • Mason KD, Carpinelli MR, Fletcher JI, Collinge JE, Hilton AA, Ellis S, Kelly PN, Ekert PG, Metcalf D, Roberts AW, Huang DC, Kile BT (2007) Programmed anuclear cell death delimits platelet life span. Cell 128:1173–1186

    Article  PubMed  CAS  Google Scholar 

  • Mohr M, Klempt M, Rathkolb B, Hrabé de Angelis M, Wolf E, Aigner B (2004) Hypercholesterolemia in ENU-induced mouse mutants. J Lipid Res 45:2132–2137

    Article  PubMed  CAS  Google Scholar 

  • Moser AR, Pitot HC, Dove WF (1990) A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 247:322–324

    Article  PubMed  CAS  Google Scholar 

  • Nicklas W, Baneux P, Boot R, Decelle T, Deeny AA, Fumanelli M, Illgen-Wilcke B (2002) Recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units. Lab Anim 36:20–42

    Article  PubMed  CAS  Google Scholar 

  • Nolan PM, Peters J, Strivens M, Rogers D, Hagan J, Spurr N, Gray IC, Vizor L, Brooker D, Whitehill E, Washbourne R, Hough T, Greenaway S, Hewitt M, Liu X, McCormack S, Pickford K, Selley R, Wells C, Tymowska-Lalanne Z, Roby P, Glenister P, Thornton C, Thaung C, Stevenson JA, Arkell R, Mburu P, Hardisty R, Kiernan A, Erven A, Steel KP, Voegeling S, Guenet JL, Nickols C, Sadri R, Nasse M, Isaacs A, Davies K, Browne M, Fisher EM, Martin J, Rastan S, Brown SD, Hunter J (2000) A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse. Nat Genet 25:440–443

    Article  PubMed  CAS  Google Scholar 

  • Rank G, Sutton R, Marshall V, Lundie RJ, Caddy J, Romeo T, Fernandez K, McCormack MP, Cooke BM, Foote SJ, Crabb BS, Curtis DJ, Hilton DJ, Kile BT, Jane SM (2009) Novel roles for erythroid Ankyrin-1 revealed through an ENU-induced null mouse mutant. Blood 113:3352–3362

    Article  PubMed  CAS  Google Scholar 

  • Rathkolb B, Decker T, Fuchs E, Soewarto D, Fella C, Heffner S, Pargent W, Wanke R, Balling R, Hrabé de Angelis M, Kolb HJ, Wolf E (2000) The clinical-chemical screen in the Munich ENU Mouse Mutagenesis Project: screening for clinically relevant phenotypes. Mamm Genome 11:543–546

    Article  PubMed  CAS  Google Scholar 

  • Sandberg ML, Sutton SE, Pletcher MT, Wiltshire T, Tarantino LM, Hogenesch JB, Cooke MP (2005) c-Myb and p300 regulate hematopoietic stem cell proliferation and differentiation. Dev Cell 8:153–166

    Article  PubMed  CAS  Google Scholar 

  • Schmidt PJ, Toran PT, Giannetti AM, Bjorkman PJ, Andrews NC (2008) The transferrin receptor modulates Hfe-dependent regulation of hepcidin expression. Cell Metab 7:205–214

    Article  PubMed  Google Scholar 

  • Soranzo N, Spector TD, Mangino M, Kuhnel B, Rendon A, Teumer A, Willenborg C, Wright B, Chen L, Li M, Salo P, Voight BF, Burns P, Laskowski RA, Xue Y, Menzel S, Altshuler D, Bradley JR, Bumpstead S, Burnett MS, Devaney J, Doring A, Elosua R, Epstein SE, Erber W, Falchi M, Garner SF, Ghori MJ, Goodall AH, Gwilliam R, Hakonarson HH, Hall AS, Hammond N, Hengstenberg C, Illig T, Konig IR, Knouff CW, McPherson R, Melander O, Mooser V, Nauck M, Nieminen MS, O’Donnell CJ, Peltonen L, Potter SC, Prokisch H, Rader DJ, Rice CM, Roberts R, Salomaa V, Sambrook J, Schreiber S, Schunkert H, Schwartz SM, Serbanovic-Canic J, Sinisalo J, Siscovick DS, Stark K, Surakka I, Stephens J, Thompson JR, Volker U, Volzke H, Watkins NA, Wells GA, Wichmann HE, Van Heel DA, Tyler-Smith C, Thein SL, Kathiresan S, Perola M, Reilly MP, Stewart AF, Erdmann J, Samani NJ, Meisinger C, Greinacher A, Deloukas P, Ouwehand WH, Gieger C (2009) A genome-wide meta-analysis identifies 22 loci associated with eight hematological parameters in the HaemGen consortium. Nat Genet 41:1182–1190

    Article  PubMed  CAS  Google Scholar 

  • Stevenson WS, Hyland CD, Zhang JG, Morgan PO, Willson TA, Gill A, Hilton AA, Viney EM, Bahlo M, Masters SL, Hennebry S, Richardson SJ, Nicola NA, Metcalf D, Hilton DJ, Roberts AW, Alexander WS (2010) Deficiency of 5-hydroxyisourate hydrolase causes hepatomegaly and hepatocellular carcinoma in mice. Proc Natl Acad Sci USA 107:16625–16630

    Article  PubMed  CAS  Google Scholar 

  • Su LK, Kinzler KW, Vogelstein B, Preisinger AC, Moser AR, Luongo C, Gould KA, Dove WF (1992) Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science 256:668–670

    Article  PubMed  CAS  Google Scholar 

  • Takahasi KR, Sakuraba Y, Gondo Y (2007) Mutational pattern and frequency of induced nucleotide changes in mouse ENU mutagenesis. BMC Mol Biol 8:52

    Article  PubMed  Google Scholar 

  • Tian M, Campagna DR, Woodward LS, Justice MJ, Fleming MD (2008) Hem6: an ENU-induced recessive hypochromic microcytic anemia mutation in the mouse. Blood 112:4308–4313

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the German Human Genome Project (DHGP) and the National Genome Research Network (NGFN) (MHdA, EW).

Disclosure

The authors have no conflicts of interest, financial or otherwise, to declare.

Author information

Authors and Affiliations

  1. Department of Veterinary Sciences, Chair for Molecular Animal Breeding and Biotechnology, and Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Moorversuchsgut, Hackerstr. 27, 85764, Oberschleiβheim, Munich, Germany

    Bernhard Aigner, Birgit Rathkolb, Martina Klempt & Eckhard Wolf

  2. Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, and Chair for Experimental Genetics, TU Munich, Freising-Weihenstephan, Helmholtz Zentrum München, Neuherberg, Germany

    Sibylle Wagner, Dian Michel, Matthias Klaften & Martin Hrabé de Angelis

  3. Ingenium Pharmaceuticals, Martinsried, Germany

    Jürgen Laufs, Boris Schneider & Reinhard Sedlmeier

Authors
  1. Bernhard Aigner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Birgit Rathkolb
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martina Klempt
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sibylle Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dian Michel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Matthias Klaften
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jürgen Laufs
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Boris Schneider
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Reinhard Sedlmeier
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Martin Hrabé de Angelis
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Eckhard Wolf
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bernhard Aigner.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Aigner, B., Rathkolb, B., Klempt, M. et al. Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in hematological parameters. Mamm Genome 22, 495–505 (2011). https://doi.org/10.1007/s00335-011-9328-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00335-011-9328-4

Keywords

Search

Navigation