Skip to main content

Mouse Models: Knockouts/Knockins

  • Chapter
  • First Online:

Abstract

There is an enormous initiative to establish the genetic basis for addictive disorders. While forward genetic studies focus on identification of risk factors and protective genes impacting on addictive behaviors of humans and animal models, much effort is devoted to functions of these genes and polymorphisms relevant to substance abuse in reverse genetic studies with gene manipulation in intact organisms. Animal models, particularly the laboratory mouse, offer attractive systems with which scientists can work toward the goal. In this chapter, we introduce the state-of-the-art gene targeting technology in reverse genetics. By using homologous recombination and a well-designed strategy, a gene of special interest in the mouse genome can be eliminated or modified for function alteration on purpose. The resultant mutant animals provide excellent models for subsequent studies of gene function in development, pathophysiology, and behaviors. Various genes relevant to addiction to a variety of drugs of abuse have been examined with knockout, knockin, and conditional gene targeting technologies. Studies in these genetic engineered animal models are starting to yield some converging findings, providing crucial insights into brain regions and genes associated with drug addiction, and speeding up our understanding of the complex neuronal processes involved in these disorders. In addition, these animal models provide important platforms for the development of prevention and intervention strategies targeted at an individual’s specific needs.

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

Buying options

Chapter
USD   29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   299.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   379.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   329.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

References

  1. Akbarian S, Bates B, Liu RJ et al (2001) Neurotrophin-3 modulates noradrenergic neuron function and opiate withdrawal. Mol Psychiatry 6:593–604

    Article  PubMed  CAS  Google Scholar 

  2. Akbarian S, Rios M, Liu RJ et al (2002) Brain-derived neurotrophic factor is essential for opiate-induced plasticity of noradrenergic neurons. J Neurosci 22:4153–4162

    PubMed  CAS  Google Scholar 

  3. Bastia E, Xu YH, Scibelli AC et al (2005) A crucial role for forebrain adenosine A(2A) receptors in amphetamine sensitization. Neuropsychopharmacology 30:891–900

    Article  PubMed  CAS  Google Scholar 

  4. Beard C, Hochedlinger K, Plath K et al (2006) Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis 44:23–28

    Article  PubMed  CAS  Google Scholar 

  5. Benavides DR, Bibb JA (2004) Role of Cdk5 in drug abuse and plasticity. Ann NY Acad Sci 1025:335–344

    Article  PubMed  CAS  Google Scholar 

  6. Benavides DR, Quinn JJ, Zhong P et al (2007) Cdk5 modulates cocaine reward, motivation, and striatal neuron excitability. J Neurosci 27:12967–12976

    Article  PubMed  CAS  Google Scholar 

  7. Bennett B, Downing C, Parker C et al (2006) Mouse genetic models in alcohol research. Trends Genet 22:367–374

    Article  PubMed  CAS  Google Scholar 

  8. Beuten J, Ma JZ, Payne TJ et al (2005) Significant association of BDNF haplotypes in European-American male smokers but not in European-American female or African-American smokers. Am J Med Genet B Neuropsychiatr Genet 139:73–80

    Google Scholar 

  9. Bierut LJ, Madden PA, Breslau N et al (2007) Novel genes identified in a high-density genome wide association study for nicotine dependence. Hum Mol Genet 16:24–35

    Article  PubMed  CAS  Google Scholar 

  10. Branda CS, Dymecki SM (2004) Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev Cell 6:7–28

    Article  PubMed  CAS  Google Scholar 

  11. Brook FA, Gardner RL (1997) The origin and efficient derivation of embryonic stem cells in the mouse. Proc Natl Acad Sci USA 94:5709–5712

    Article  PubMed  CAS  Google Scholar 

  12. Bueller JA, Aftab M, Sen S et al (2006) BDNF Val66Met allele is associated with reduced hippocampal volume in healthy subjects. Biol Psychiatry 59:812–815

    Article  PubMed  CAS  Google Scholar 

  13. Chen ZY, Jing D, Bath KG et al (2006) Genetic variant BDNF (Val66Met) polymorphism alters anxiety-related behavior. Science 314:140–143

    Article  PubMed  CAS  Google Scholar 

  14. Chen ZY, Patel PD, Sant G et al (2004) Variant brain-derived neurotrophic factor (BDNF) (Met66) alters the intracellular trafficking and activity-dependent secretion of wild-type BDNF in neurosecretory cells and cortical neurons. J Neurosci 24:4401–4411

    Article  PubMed  CAS  Google Scholar 

  15. Cheng CY, Hong CJ, Yu YW et al (2005) Brain-derived neurotrophic factor (Val66Met) genetic polymorphism is associated with substance abuse in males. Brain Res Mol Brain Res 140:86–90

    Article  PubMed  CAS  Google Scholar 

  16. Chung Y, Klimanskaya I, Becker S et al (2006) Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres. Nature 439:216–219

    Article  PubMed  CAS  Google Scholar 

  17. Churchill GA, Airey DC, Allayee H et al (2004) The collaborative cross, a community resource for the genetic analysis of complex traits. Nat Genet 36:1133–1137

    Article  PubMed  CAS  Google Scholar 

  18. Conover JC, Yancopoulos GD (1997) Neurotrophin regulation of the developing nervous system: analyses of knockout mice. Rev Neurosci 8:13–27

    PubMed  CAS  Google Scholar 

  19. Crabbe JC, Phillips TJ, Harris RA et al (2006) Alcohol-related genes: contributions from studies with genetically engineered mice. Addict Biol 11:195–269

    Article  PubMed  CAS  Google Scholar 

  20. Deroche-Gamonet V, Sillaber I, Aouizerate B et al (2003) The glucocorticoid receptor as a potential target to reduce cocaine abuse. J Neurosci 23:4785–4790

    PubMed  CAS  Google Scholar 

  21. Doetschman T, Gregg RG, Maeda N et al (1987) Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330:576–578

    Article  PubMed  CAS  Google Scholar 

  22. Egan MF, Kojima M, Callicott JH et al (2003) The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell 112:257–269

    Article  PubMed  CAS  Google Scholar 

  23. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156

    Article  PubMed  CAS  Google Scholar 

  24. Feil R, Brocard J, Mascrez B et al (1996) Ligand-activated site-specific recombination in mice. Proc Natl Acad Sci USA 93:10887–10890

    Article  PubMed  CAS  Google Scholar 

  25. Feng Y, Niu T, Xing H et al (2004) A common haplotype of the nicotine acetylcholine receptor alpha 4 subunit gene is associated with vulnerability to nicotine addiction in men. Am J Hum Genet 75:112–121

    Article  PubMed  CAS  Google Scholar 

  26. Gaveriaux-Ruff C, Kieffer BL (2007) Conditional gene targeting in the mouse nervous system: insights into brain function and diseases. Pharmacol Ther 113:619–634

    Article  PubMed  CAS  Google Scholar 

  27. Giros B, Jaber M, Jones SR et al (1996) Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379:606–612

    Article  PubMed  CAS  Google Scholar 

  28. Goldman D, Oroszi G, Ducci F (2005) The genetics of addictions: uncovering the genes. Nat Rev Genet 6:521–532

    Article  PubMed  CAS  Google Scholar 

  29. Gordon JW, Scangos GA, Plotkin DJ et al (1980) Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA 77:7380–7384

    Article  PubMed  CAS  Google Scholar 

  30. Graham DL, Edwards S, Bachtell RK et al (2007) Dynamic BDNF activity in nucleus accumbens with cocaine use increases self-administration and relapse. Nat Neurosci 10:1029–1037

    Article  PubMed  CAS  Google Scholar 

  31. Gratacos M, Gonzalez JR, Mercader JM et al (2007) Brain-derived neurotrophic factor Val66Met and psychiatric disorders: meta-analysis of case-control studies confirm association to substance-related disorders, eating disorders, and schizophrenia. Biol Psychiatry 61:911–922

    Article  PubMed  CAS  Google Scholar 

  32. Grimm JW, Lu L, Hayashi T et al (2003) Time-dependent increases in brain-derived neurotrophic factor protein levels within the mesolimbic dopamine system after withdrawal from cocaine: implications for incubation of cocaine craving. J Neurosci 23:742–747

    PubMed  CAS  Google Scholar 

  33. Hall FS, Drgonova J, Goeb M et al (2003) Reduced behavioral effects of cocaine in heterozygous brain-derived neurotrophic factor (BDNF) knockout mice. Neuropsychopharmacology 28:1485–1490

    Article  PubMed  CAS  Google Scholar 

  34. Hall FS, Li XF, Sora I et al (2002) Cocaine mechanisms: enhanced cocaine, fluoxetine and nisoxetine place preferences following monoamine transporter deletions. Neuroscience 115:153–161

    Article  PubMed  CAS  Google Scholar 

  35. Hasty P, Ramirez-Solis R, Krumlauf R et al (1991) Introduction of a subtle mutation into the Hox-2.6 locus in embryonic stem cells. Nature 350:243–246

    Article  PubMed  CAS  Google Scholar 

  36. Hawasli AH, Benavides DR, Nguyen C et al (2007) Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation. Nat Neurosci 10:880–886

    Article  PubMed  CAS  Google Scholar 

  37. Horger BA, Iyasere CA, Berhow MT et al (1999) Enhancement of locomotor activity and conditioned reward to cocaine by brain-derived neurotrophic factor. J Neurosci 19:4110–4122

    PubMed  CAS  Google Scholar 

  38. Itoh K, Hashimoto K, Shimizu E et al (2005) Association study between brain-derived neurotrophic factor gene polymorphisms and methamphetamine abusers in Japan. Am J Med Genet B Neuropsychiatr Genet 132B:70–73

    Article  PubMed  Google Scholar 

  39. Jiang X, Xu K, Hoberman J et al (2005) BDNF variation and mood disorders: a novel functional promoter polymorphism and Val66Met are associated with anxiety but have opposing effects. Neuropsychopharmacology 30:1353–1361

    PubMed  CAS  Google Scholar 

  40. Joyner AL (2000) Gene targeting: a practical approach, 2nd edn. University Press, Oxford, New York

    Google Scholar 

  41. Kuehn MR, Bradley A, Robertson EJ et al (1987) A potential animal model for Lesch-Nyhan syndrome through introduction of HPRT mutations into mice. Nature 326:295–298

    Article  PubMed  CAS  Google Scholar 

  42. Labarca C, Schwarz J, Deshpande P et al (2001) Point mutant mice with hypersensitive alpha 4 nicotinic receptors show dopaminergic deficits and increased anxiety. Proc Natl Acad Sci USA 98:2786–2791

    Article  PubMed  CAS  Google Scholar 

  43. Lakso M, Sauer B, Mosinger B Jr et al (1992) Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci USA 89:6232–6236

    Article  PubMed  CAS  Google Scholar 

  44. Lang UE, Sander T, Lohoff FW et al (2007) Association of the met66 allele of brain-derived neurotrophic factor (BDNF) with smoking. Psychopharmacology (Berl) 190:433–439

    Article  CAS  Google Scholar 

  45. Li MD, Beuten J, Ma JZ et al (2005) Ethnic- and gender-specific association of the nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4) with nicotine dependence. Hum Mol Genet 14:1211–1219

    Article  PubMed  CAS  Google Scholar 

  46. Liu QR, Walther D, Drgon T et al (2005) Human brain derived neurotrophic factor (BDNF) genes, splicing patterns, and assessments of associations with substance abuse and Parkinson’s Disease. Am J Med Genet B Neuropsychiatr Genet 134B:93–103

    Article  PubMed  Google Scholar 

  47. Lu B (2003) Pro-region of neurotrophins: role in synaptic modulation. Neuron 39:735–738

    Article  PubMed  CAS  Google Scholar 

  48. 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

    Article  PubMed  CAS  Google Scholar 

  49. Marubio LM, Gardier AM, Durier S et al (2003) Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptors. Eur J Neurosci 17:1329–1337

    Article  PubMed  CAS  Google Scholar 

  50. Matsui Y, Zsebo K, Hogan BL (1992) Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70:841–847

    Article  PubMed  CAS  Google Scholar 

  51. Matsushita S, Kimura M, Miyakawa T et al (2004) Association study of brain-derived neurotrophic factor gene polymorphism and alcoholism. Alcohol Clin Exp Res 28:1609–1612

    Article  PubMed  CAS  Google Scholar 

  52. McClung CA (2007) Circadian rhythms, the mesolimbic dopaminergic circuit, and drug addiction. Sci World J 7:194–202

    Article  Google Scholar 

  53. Miyakawa T, Leiter LM, Gerber DJ et al (2003) Conditional calcineurin knockout mice exhibit multiple abnormal behaviors related to schizophrenia. Proc Natl Acad Sci USA 100:8987–8992

    Article  PubMed  CAS  Google Scholar 

  54. Nestler EJ (2000) Genes and addiction. Nat Genet 26:277–281

    Article  PubMed  CAS  Google Scholar 

  55. Nielsen DA, Ji F, Yuferov V et al (2008) Genotype patterns that contribute to increased risk for or protection from developing heroin addiction. Mol Psychiatry 13:417–428

    Article  PubMed  CAS  Google Scholar 

  56. Palmiter RD, Sandgren EP, Avarbock MR et al (1991) Heterologous introns can enhance expression of transgenes in mice. Proc Natl Acad Sci USA 88:478–482

    Article  PubMed  CAS  Google Scholar 

  57. Pezawas L, Verchinski BA, Mattay VS et al (2004) The brain-derived neurotrophic factor val66met polymorphism and variation in human cortical morphology. J Neurosci 24:10099–10102

    Article  PubMed  CAS  Google Scholar 

  58. Phillips TJ, Hen R, Crabbe JC (1999) Complications associated with genetic background effects in research using knockout mice. Psychopharmacology (Berl) 147:5–7

    Article  CAS  Google Scholar 

  59. Phillips TJ, Kamens HM, Wheeler JM (2008) Behavioral genetic contributions to the study of addiction-related amphetamine effects. Neurosci Biobehav Rev 32:707–759

    Article  PubMed  CAS  Google Scholar 

  60. Picciotto MR, Zoli M, Rimondini R et al (1998) Acetylcholine receptors containing the beta2 subunit are involved in the reinforcing properties of nicotine. Nature 391:173–177

    Article  PubMed  CAS  Google Scholar 

  61. Recillas-Targa F, Valadez-Graham V, Farrell CM (2004) Prospects and implications of using chromatin insulators in gene therapy and transgenesis. Bioessays 26:796–807

    Article  PubMed  CAS  Google Scholar 

  62. Rocha BA, Fumagalli F, Gainetdinov RR et al (1998) Cocaine self-administration in dopamine-transporter knockout mice. Nat Neurosci 1:132–137

    Article  PubMed  CAS  Google Scholar 

  63. Silverman MA, Neale MC, Sullivan PF et al (2000) Haplotypes of four novel single nucleotide polymorphisms in the nicotinic acetylcholine receptor beta2-subunit (CHRNB2) gene show no association with smoking initiation or nicotine dependence. Am J Med Genet 96:646–653

    Article  PubMed  CAS  Google Scholar 

  64. Sora I, Hall FS, Andrews AM et al (2001) Molecular mechanisms of cocaine reward: combined dopamine and serotonin transporter knockouts eliminate cocaine place preference. Proc Natl Acad Sci USA 98:5300–5305

    Article  PubMed  CAS  Google Scholar 

  65. Sora I, Wichems C, Takahashi N et al (1998) Cocaine reward models: conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice. Proc Natl Acad Sci USA 95:7699–7704

    Article  PubMed  CAS  Google Scholar 

  66. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71

    Article  PubMed  CAS  Google Scholar 

  67. Stacey A, Schnieke A, McWhir J et al (1994) Use of double-replacement gene targeting to replace the murine alpha-lactalbumin gene with its human counterpart in embryonic stem cells and mice. Mol Cell Biol 14:1009–1016

    PubMed  CAS  Google Scholar 

  68. Stec DE, Morimoto S, Sigmund CD (2001) Vectors for high-level expression of cDNAs controlled by tissue-specific promoters in transgenic mice. Biotechniques 31:256–258, 260

    PubMed  CAS  Google Scholar 

  69. Surtees PG, Wainwright NW, Willis-Owen SA et al (2007) No association between the BDNF Val66Met polymorphism and mood status in a non-clinical community sample of 7389 older adults. J Psychiatr Res 41:404–409

    Article  PubMed  Google Scholar 

  70. Szeszko PR, Lipsky R, Mentschel C et al (2005) Brain-derived neurotrophic factor val66met polymorphism and volume of the hippocampal formation. Mol Psychiatry 10:631–636

    Article  PubMed  CAS  Google Scholar 

  71. Tapper AR, McKinney SL, Nashmi R et al (2004) Nicotine activation of alpha4* receptors: sufficient for reward, tolerance, and sensitization. Science 306:1029–1032

    Article  PubMed  CAS  Google Scholar 

  72. Tesar PJ (2005) Derivation of germ-line-competent embryonic stem cell lines from preblastocyst mouse embryos. Proc Natl Acad Sci USA 102:8239–8244

    Article  PubMed  CAS  Google Scholar 

  73. Thomas KR, Capecchi MR (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503–512

    Article  PubMed  CAS  Google Scholar 

  74. Touw K, Hoggatt AM, Simon G et al (2007) Hprt-targeted transgenes provide new insights into smooth muscle-restricted promoter activity. Am J Physiol Cell Physiol 292:C1024–C1032

    Article  PubMed  CAS  Google Scholar 

  75. Tsai SJ, Liao DL, Yu YW et al (2005) A study of the association of (Val66Met) polymorphism in the brain-derived neurotrophic factor gene with alcohol dependence and extreme violence in Chinese males. Neurosci Lett 381:340–343

    Article  PubMed  CAS  Google Scholar 

  76. Tymms MJ, Kola I (2001) Gene knockout protocols. Humana, Totowa, NJ

    Book  Google Scholar 

  77. Uhl GR, Drgon T, Johnson C et al (2008a) “Higher order” addiction molecular genetics: convergent data from genome-wide association in humans and mice. Biochem Pharmacol 75:98–111

    Article  PubMed  CAS  Google Scholar 

  78. Uhl GR, Drgon T, Liu QR et al (2008b) Genome-wide association for methamphetamine dependence: convergent results from 2 samples. Arch Gen Psychiatry 65:345–355

    Article  PubMed  CAS  Google Scholar 

  79. Uhl GR, Liu QR, Drgon T et al (2007) Molecular genetics of nicotine dependence and abstinence: whole genome association using 520,000 SNPs. BMC Genet 8:10

    Article  PubMed  Google Scholar 

  80. Valverde O, Mantamadiotis T, Torrecilla M et al (2004) Modulation of anxiety-like behavior and morphine dependence in CREB-deficient mice. Neuropsychopharmacology 29:1122–1133

    Article  PubMed  CAS  Google Scholar 

  81. Wu H, Liu X, Jaenisch R (1994) Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells. Proc Natl Acad Sci USA 91:2819–2823

    Article  PubMed  CAS  Google Scholar 

  82. Xu F, Gainetdinov RR, Wetsel WC et al (2000) Mice lacking the norepinephrine transporter are supersensitive to psychostimulants. Nat Neurosci 3:465–471

    Article  PubMed  CAS  Google Scholar 

  83. Zhang H, Ozbay F, Lappalainen J et al (2006) Brain derived neurotrophic factor (BDNF) gene variants and Alzheimer’s disease, affective disorders, posttraumatic stress disorder, schizophrenia, and substance dependence. Am J Med Genet B Neuropsychiatr Genet 141B:387–393

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The preparation of this book chapter was supported in part by National Institutes of Health grants DA-12844 and DA-13783 to Ming D. Li.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ming D. Li .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media, LLC

About this chapter

Cite this chapter

Huang, W., Xu, W., Li, M.D. (2010). Mouse Models: Knockouts/Knockins. In: Johnson, B. (eds) Addiction Medicine. Springer, New York, NY. https://doi.org/10.1007/978-1-4419-0338-9_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4419-0338-9_9

  • Published:

  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4419-0337-2

  • Online ISBN: 978-1-4419-0338-9

  • eBook Packages: MedicineMedicine (R0)

Publish with us

Policies and ethics