Overview of Designing Genetically Engineered Mouse (GEM) Models

  • Thomas Doetschman
  • L. Philip Sanford


It is very important to spend time and effort on vector design considerations when planning to make a GEM. The vector designer will ask what information is desired from the genetically modified animal, and an engineering scheme will be devised. It is strongly recommended that the investigator consult experienced GEM vector producers with all the information that is desired from the GEM. The investigator will be apprised of the feasibility of each design consideration, and usually learns of additional design elements that may expand the information that can be obtained from the GEM and that can in turn expand the overall research yield. Our experience is that the extra time, effort, and care that is put into the coordination of GEM design with research objectives saves much time and effort in the long run. In addition, we have found that careful GEM design consideration will greatly improve the success of GEM production. In this chapter, we discuss gene targeting design considerations that should be made before initiating production of the engineered mouse strain.


Selectable Marker Gene loxP Site DHFR Gene Selection Cassette Advance Intercross Line 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Almind K, Kulkarni RN, Lannon SM, Kahn CR (2003) Identification of interactive loci linked to insulin and leptin in mice with genetic insulin resistance. Diabetes 52:1535–1543PubMedCrossRefGoogle Scholar
  2. Antoch MP, Song EJ, Chang AM, Vitaterna MH, Zhao Y, Wilsbacher LD, Sangoram AM, King DP, Pinto LH, Takahashi JS (1997) Functional identification of the mouse circadian Clock gene by transgenic BAC rescue. Cell 89:655–667PubMedCrossRefGoogle Scholar
  3. Askew GR, Doetschman T, Lingrel JB (1993) Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy. Mol Cell Biol 13:4115–4124PubMedGoogle Scholar
  4. Bhattacharyya R, Bhaumik M, Raju TS, Stanley P (2002) Truncated, inactive N-acetylglucosa-minyltransferase III (GlcNAc-TIII) induces neurological and other traits absent in mice that lack GlcNAc-TIII. J Biol Chem 277:26300–26309PubMedCrossRefGoogle Scholar
  5. Bradley A, Evans M, Kaufman MH, Robertson E (1984) Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309:255–256PubMedCrossRefGoogle Scholar
  6. Branda CS, Dymecki SM (2004) Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev Cell 6:7–28PubMedCrossRefGoogle Scholar
  7. Brinster RL (1974) The effect of cells transferred into the mouse blastocyst on subsequent development. J Exp Med 140:1049–1056PubMedCrossRefGoogle Scholar
  8. Carver BS, Pandolfi PP (2006) Mouse modeling in oncologic preclinical and translational research. Clin Cancer Res 12:5305–5311PubMedCrossRefGoogle Scholar
  9. Copeland NG, Jenkins NA, Court DL (2001) Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet 2:769–779PubMedCrossRefGoogle Scholar
  10. Cossee M, Puccio H, Gansmuller A, Koutnikova H, Dierich A, LeMeur M, Fischbeck K, Dolle P, Koenig M (2000) Inactivation of the Friedreich ataxia mouse gene leads to early embryonic lethality without iron accumulation. Hum Mol Genet 9:1219–1226PubMedCrossRefGoogle Scholar
  11. Court DL, Sawitzke JA, Thomason LC (2002) Genetic engineering using homologous recombination. Annu Rev Genet 36:361–388PubMedCrossRefGoogle Scholar
  12. Daniel D, Chiu C, Giraudo E, Inoue M, Mizzen LA, Chu NR, Hanahan D (2005) CD4+ T cell-mediated antigen-specific immunotherapy in a mouse model of cervical cancer. Cancer Res 65:2018–2025PubMedCrossRefGoogle Scholar
  13. Dekker M, Brouwers C, te-Riele H (2003) Targeted gene modification in mismatch-repair-deficient embryonic stem cells by single-stranded DNA oligonucleotides. Nucleic Acids Res 31:e27PubMedCrossRefGoogle Scholar
  14. Den Z, Cheng X, Merched-Sauvage M, Koch PJ (2006) Desmocollin 3 is required for pre-implantation development of the mouse embryo. J Cell Sci 119:482–489PubMedCrossRefGoogle Scholar
  15. Deng C, Capecchi MR (1992) Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol Cell Biol 12:3365–3371PubMedGoogle Scholar
  16. Doetschman T, Gregg RG, Maeda N, Hooper ML, Melton DW, Thompson S, Smithies O (1987) Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330:576–578PubMedCrossRefGoogle Scholar
  17. Doetschman T, Maeda N, Smithies O (1988) Targeted mutation of the Hprt gene in mouse embryonic stem cells. Proc Natl Acad Sci USA 85:8583–8587PubMedCrossRefGoogle Scholar
  18. Doetschman T (1994) Gene transfer in embryonic stem cells. In: Pinkert CA (ed) Transgenic animal technology: a laboratory handbook. Academic, New York, pp 115–146Google Scholar
  19. Doetschman T (1999) Interpretation of phenotype in genetically engineered mice. Lab Anim Sci 49:137–143PubMedGoogle Scholar
  20. Evans MJ (1972) The isolation and properties of a clonal tissue culture strain of pluripotent mouse teratoma cells. J Embryol Exp Morphol 28:163–176PubMedGoogle Scholar
  21. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156PubMedCrossRefGoogle Scholar
  22. Giraudo E, Inoue M, Hanahan D (2004) An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis. J Clin Invest 114:623–633PubMedGoogle Scholar
  23. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH (1980) Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA 77:7380–7384PubMedCrossRefGoogle Scholar
  24. Gossler A, Doetschman T, Korn R, Serfling E, Kemler R (1986) Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc Natl Acad Sci USA 83:9065–9069PubMedCrossRefGoogle Scholar
  25. Grosveld F, van Assendelft GB, Greaves DR, Kollias G (1987) Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell 51:975–985PubMedCrossRefGoogle Scholar
  26. Hanahan D (1989) Transgenic mice as probes into complex systems. Science 246:1265–1275PubMedCrossRefGoogle Scholar
  27. Hasty P, Ramirez-Solis R, Krumlauf R, Bradley A (1991a) Introduction of a subtle mutation into the Hox-2.6 locus in embryonic stem cells. Nature 350:243–246PubMedCrossRefGoogle Scholar
  28. Hasty P, Rivera-Perez J, Bradley A (1991b) The length of homology required for gene targeting in embryonic stem cells. Mol Cell Biol 11:5586–5591PubMedGoogle Scholar
  29. Hide T, Hatakeyama J, Kimura-Yoshida C, Tian E, Takeda N, Ushio Y, Shiroishi T, Aizawa S, Matsuo I (2002) Genetic modifiers of otocephalic phenotypes in Otx2 heterozygous mutant mice. Development 129:4347–4357PubMedGoogle Scholar
  30. Hooper M, Hardy K, Handyside A, Hunter S, Monk M (1987) HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature 326:292–295PubMedCrossRefGoogle Scholar
  31. Huang LS, Voyiaziakis E, Markenson DF, Sokol KA, Hayek T, Breslow JL (1995) apo B gene knockout in mice results in embryonic lethality in homozygotes and neural tube defects, male infertility, and reduced HDL cholesterol ester and apo A-I transport rates in heterozygotes. J Clin Invest 96:2152–2161PubMedCrossRefGoogle Scholar
  32. Huang PL, Dawson TM, Bredt DS, Snyder SH, Fishman MC (1993) Targeted disruption of the neuronal nitric oxide synthase gene. Cell 75:1273–1286PubMedCrossRefGoogle Scholar
  33. Hung KE, Faca V, Song K, Sarracino DA, Richard LG, Krastins B, Forrester S, Porter A, Kunin A, Mahmood U, Haab BB, Hanash SM, Kucherlapati R (2009) Comprehensive proteome analysis of an Apc mouse model uncovers proteins associated with intestinal tumorigenesis. Cancer Prev Res 2:224–233CrossRefGoogle Scholar
  34. Jackson-Grusby L (2002) Modeling cancer in mice. Oncogene 21:5504–5514PubMedCrossRefGoogle Scholar
  35. Jaenisch R (1976) Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc Natl Acad Sci USA 73:1260–1264PubMedCrossRefGoogle Scholar
  36. Jaenisch R (1988) Transgenic animals. Science 240:1468–1474PubMedCrossRefGoogle Scholar
  37. Jaenisch R, Mintz B (1974) Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA. Proc Natl Acad Sci USA 71:1250–1254PubMedCrossRefGoogle Scholar
  38. Jiang W, Anderson MS, Bronson R, Mathis D, Benoist C (2005) Modifier loci condition autoimmunity provoked by Aire deficiency. J Exp Med 202:805–815PubMedCrossRefGoogle Scholar
  39. Johnson RS, Sheng M, Greenberg ME, Kolodner RD, Papaioannou VE, Spiegelman BM (1989) Targeting of nonexpressed genes in embryonic stem cells via homologous recombination. Science 245:1234–1236PubMedCrossRefGoogle Scholar
  40. Jonkers J, Berns A (2002) Conditional mouse models of sporadic cancer. Nat Rev Cancer 2:251–265PubMedCrossRefGoogle Scholar
  41. Jung S, Rajewsky K, Radbruch A (1993) Shutdown of class switch recombination by deletion of a switch region control element. Science 259:984–987PubMedCrossRefGoogle Scholar
  42. Kallapur S, Ormsby I, Doetschman T (1999) Strain dependency of TGFbeta1 function during embryogenesis. Mol Reprod Dev 52:341–349PubMedCrossRefGoogle Scholar
  43. Koller BH, Hagemann LJ, Doetschman T, Hagaman JR, Huang S, Williams PJ, First NL, Maeda N, Smithies O (1989) Germ-line transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells. Proc Natl Acad Sci USA 86:8927–8931PubMedCrossRefGoogle Scholar
  44. Koller BH, Smithies O (1992) Altering genes in animals by gene targeting. Annu Rev Immunol 10:705–730PubMedCrossRefGoogle Scholar
  45. Kuhn R, Schwenk F, Aguet M, Rajewsky K (1995) Inducible gene targeting in mice. Science 269:1427–1429PubMedCrossRefGoogle Scholar
  46. Lamb BT, Sisodia SS, Lawler AM, Slunt HH, Kitt CA, Kearns WG, Pearson PL, Price DL, Gearhart JD (1993) Introduction and expression of the 400 kilobase amyloid precursor protein gene in transgenic mice. Nat Genet 5:22–30PubMedCrossRefGoogle Scholar
  47. Mansour SL, Thomas KR, Capecchi MR (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336:348–352PubMedCrossRefGoogle Scholar
  48. Mansour SL (1990) Gene targeting in murine embryonic stem cells: introduction of specific alterations into the mammalian genome. Genet Anal Tech Appl 7:219–227PubMedCrossRefGoogle Scholar
  49. 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 78:7634–7638PubMedCrossRefGoogle Scholar
  50. McLin JP, Steward O (2006) Comparison of seizure phenotype and neurodegeneration induced by systemic kainic acid in inbred, outbred, and hybrid mouse strains. Eur J Neurosci 24:2191–2202PubMedCrossRefGoogle Scholar
  51. Moens CB, Auerback AB, Conlon RA, Joyner AL, Rossant J (1992) A targeted mutation reveals a role for N-myc in branching morphogenesis in the embryonic mouse lung. Genes Develop 6:691–704Google Scholar
  52. Moll UM, Slade N (2004) p63 and p73: roles in development and tumor formation. Mol Cancer Res 2:371–386PubMedGoogle Scholar
  53. Muyrers JP, Zhang Y, Stewart AF (2001) Techniques: Recombinogenic engineering – new options for cloning and manipulating DNA. Trends Biochem Sci 26:325–331PubMedCrossRefGoogle Scholar
  54. Muyrers JP, Zhang Y, Testa G, Stewart AF (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res 27:1555–1557PubMedCrossRefGoogle Scholar
  55. Papaioannou VE, McBurney MW, Gardner RL, Evans MJ (1975) Fate of teratocarcinoma cells injected into early mouse embryos. Nature 258:70–73PubMedCrossRefGoogle Scholar
  56. Pham CT, MacIvor DM, Hug BA, Heusel JW, Ley TJ (1996) Long-range disruption of gene expression by a selectable marker cassette. Proc Natl Acad Sci USA 93:13090–13095PubMedCrossRefGoogle Scholar
  57. Rajewsky K, Gu H, Kuhn R, Betz UA, Muller W, Roes J, Schwenk F (1996) Conditional gene targeting. J Clin Invest 98:600–603PubMedCrossRefGoogle Scholar
  58. Robertson E, Bradley A, Kuehn M, Evans M (1986) Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 323:445–448PubMedCrossRefGoogle Scholar
  59. Sanford LP, Kallapur S, Ormsby I, Doetschman T (2001) Influence of genetic background on knockout mouse phenotypes. Methods Mol Biol 158:217–225PubMedGoogle Scholar
  60. Seidl KJ, Bottaro A, Vo A, Zhang J, Davidson L, Alt FW (1998) An expressed neo(r) cassette provides required functions of the 1gamma2b exon for class switching. Int Immunol 10:1683–1692PubMedCrossRefGoogle Scholar
  61. Shaw AT, Kirsch DG, Jacks T (2005) Future of early detection of lung cancer: the role of mouse models. Clin Cancer Res 11:4999s–5003sPubMedCrossRefGoogle Scholar
  62. Simpson EM, Linder CC, Sargent EE, Davisson MT, Mobraaten LE, Sharp JJ (1997) Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice. Nat Genet 16:19–27PubMedCrossRefGoogle Scholar
  63. Stevens LC (1960) Embryonic potency of embryoid bodies derived from a transplantable testicular teratoma of the mouse. Dev Biol 2:285–297PubMedCrossRefGoogle Scholar
  64. Stevens LC, Little CC (1954) Spontaneous testicular teratomas in an inbred strain of mice. Proc Natl Acad Sci USA 40:1080–1087PubMedCrossRefGoogle Scholar
  65. Sun T, Storb U (2001) Insertion of phosphoglycerine kinase (PGK)-neo 5′ of Jlambda1 dramatically enhances VJlambda1 rearrangement. J Exp Med 193:699–712PubMedCrossRefGoogle Scholar
  66. Thomas KR, Capecchi MR (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503–512PubMedCrossRefGoogle Scholar
  67. Thompson S, Clarke AR, Pow AM, Hooper ML, Melton DW (1989) Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells. Cell 56:313–321PubMedCrossRefGoogle Scholar
  68. Torres RM, Kühn R (1997) Laboratory protocols for conditional gene targeting. Oxford University Press, OxfordGoogle Scholar
  69. Tuveson DA, Jacks T (2002) Technologically advanced cancer modeling in mice. Curr Opin Genet Dev 12:105–110PubMedCrossRefGoogle Scholar
  70. Valancius V, Smithies O (1991) Testing an “in-out” targeting procedure for making subtle genomic modifications in mouse embryonic stem cells. Mol Cell Biol 11:1402–1408PubMedGoogle Scholar
  71. van Deursen J, Wieringa B (1992) Targeting of the creatine kinase M gene in embryonic stem cells using isogenic and nonisogenic vectors. Nucleic Acids Res 20:3815–3820PubMedCrossRefGoogle Scholar
  72. Van Dyke T, Jacks T (2002) Cancer modeling in the modern era: progress and challenges. Cell 108:135–144PubMedCrossRefGoogle Scholar
  73. Vazquez JC, Nogues C, Rucker EB, Piedrahita JA (1998) Factors affecting the efficiency of introducing precise genetic changes in ES cells by homologous recombination: tag-and-exchange versus the Cre-loxp system. Transgenic Res 7:181–193PubMedCrossRefGoogle Scholar
  74. Wagner TE, Hoppe PC, Jollick JD, Scholl DR, Hodinka RL, Gault JB (1981) Microinjection of a rabbit beta-globin gene into zygotes and its subsequent expression in adult mice and their ­offspring. Proc Natl Acad Sci USA 78:6376–6380PubMedCrossRefGoogle Scholar
  75. Wang J, Sarov M, Rientjes J, Fu J, Hollak H, Kranz H, Xie W, Stewart AF, Zhang Y (2006) An improved recombineering approach by adding RecA to lambda Red recombination. Mol Biotechnol 32:43–53Google Scholar
  76. Zhang H, Hasty P, Bradley A (1994) Targeting frequency for deletion vectors in embryonic stem cells. Mol Cell Biol 14:2404–2410PubMedGoogle Scholar
  77. Zheng H, Wilson JH (1990) Gene targeting in normal and amplified cell lines. Nature 344:170–173PubMedCrossRefGoogle Scholar
  78. Zhou L, Rowley DL, Mi QS, Sefcovic N, Matthes HW, Kieffer BL, Donovan DM (2001) Murine inter-strain polymorphisms alter gene targeting frequencies at the mu opioid receptor locus in embryonic stem cells. Mamm Genome 12:772–778PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Cellular and Molecular MedicineUniversity of ArizonaTucsonUSA
  2. 2.BIO5 InstituteUniversity of ArizonaTucsonUSA

Personalised recommendations