Skip to main content
SpringerLink
Log in

Generation of Rare Human NMDA Receptor Variants in Mice

  • Protocol
  • First Online:
NMDA Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2799))

  • 280 Accesses

Abstract

The analysis of rare NMDAR gene variants in mice, coupled with a fundamental understanding of NMDAR function, plays a crucial role in achieving therapeutic success when addressing NMDAR dysfunctions in human patients. For the generation of such NMDAR mouse models, a basic knowledge of receptor structure, along with skills in database sequence analysis, cloning in E. coli, genetic manipulation of embryonic stem (ES) cells, and ultimately the genetic modification of mouse embryos, is essential. Primarily, this chapter will focus on the design and synthesis of NMDAR gene-targeting vectors that can be used successfully for the genetic manipulation of mice. We will outline the core principles of the design and synthesis of a gene targeting vector that facilitates the introduction of single-point mutations in NMDAR-encoding genes in mice. The transformation of ES cells, selection of positive ES cell colonies, manipulation of mouse embryos, and genotyping strategies will be described briefly.

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

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 219.99
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

Institutional subscriptions

References

  1. Amin JB, Moody GR, Wollmuth LP (2021) From bedside-to-bench: what disease-associated variants are teaching us about the NMDA receptor. J Physiol 599(2):397–416. https://doi.org/10.1113/JP278705

    Article  CAS  PubMed  Google Scholar 

  2. Benke TA, Park K, Krey I, Camp CR, Song R, Ramsey AJ, Yuan H, Traynelis SF, Lemke J (2021) Clinical and therapeutic significance of genetic variation in the GRIN gene family encoding NMDARs. Neuropharmacology 199:108805. https://doi.org/10.1016/j.neuropharm.2021.108805

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Burnashev N, Szepetowski P (2015) NMDA receptor subunit mutations in neurodevelopmental disorders. Curr Opin Pharmacol 20:73–82. https://doi.org/10.1016/j.coph.2014.11.008

    Article  CAS  PubMed  Google Scholar 

  4. XiangWei W, Jiang Y, Yuan H (2018) De novo mutations and rare variants occurring in NMDA receptors. Curr Opin Physio 2:27–35. https://doi.org/10.1016/j.cophys.2017.12.013

    Article  Google Scholar 

  5. Burnashev N, Schoepfer R, Monyer H, Ruppersberg JP, Gunther W, Seeburg PH, Sakmann B (1992) Control by asparagine residues of calcium permeability and magnesium blockade in the NMDA receptor. Science 257(5075):1415–1419. https://doi.org/10.1126/science.1382314

    Article  CAS  PubMed  Google Scholar 

  6. Single FN, Rozov A, Burnashev N, Zimmermann F, Hanley DF, Forrest D, Curran T, Jensen V, Hvalby O, Sprengel R, Seeburg PH (2000) Dysfunctions in mice by NMDA receptor point mutations NR1(N598Q) and NR1(N598R). J Neurosci 20(7):2558–2566. https://doi.org/10.1523/JNEUROSCI.20-07-02558.2000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278. https://doi.org/10.1016/j.cell.2014.05.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Amador A, Bostick CD, Olson H, Peters J, Camp CR, Krizay D, Chen W, Han W, Tang W, Kanber A, Kim S, Teoh J, Sah M, Petri S, Paek H, Kim A, Lutz CM, Yang M, Myers SJ, Bhattacharya S, Yuan H, Goldstein DB, Poduri A, Boland MJ, Traynelis SF, Frankel WN (2020) Modelling and treating GRIN2A developmental and epileptic encephalopathy in mice. Brain 143(7):2039–2057. https://doi.org/10.1093/brain/awaa147

    Article  PubMed  PubMed Central  Google Scholar 

  9. Joyner AL (2003) Gene targeting: a practical approach, 2nd edn. Oxford University Press, Oxford

    Google Scholar 

  10. Harris NL, Senapathy P (1990) Distribution and consensus of branch point signals in eukaryotic genes: a computerized statistical analysis. Nucleic Acids Res 18(10):3015–3019. https://doi.org/10.1093/nar/18.10.3015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Gao K, Masuda A, Matsuura T, Ohno K (2008) Human branch point consensus sequence is yUnAy. Nucleic Acids Res 36(7):2257–2267. https://doi.org/10.1093/nar/gkn073

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yenofsky RL, Fine M, Pellow JW (1990) A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc Natl Acad Sci USA 87(9):3435–3439. https://doi.org/10.1073/pnas.87.9.3435

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Friedel RH (2009) Targeting embryonic stem cells. In: Cartwright EJ (ed) Transgenesis techniques. Methods in molecular biology, vol 561. Humana Press, New York, pp 185–197. https://doi.org/10.1007/978-1-60327-019-9_12

    Chapter  Google Scholar 

  14. Barbaric I, Dear TN (2009) Culture of murine embryonic stem cells. In: Cartwright EJ (ed) Transgenesis techniques. Methods in molecular biology, vol 561. Humana Press, New York, pp 161–184. https://doi.org/10.1007/978-1-60327-019-9_11

    Chapter  Google Scholar 

  15. Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  16. Bronstein I, Voyta JC, Murphy OJ, Tizard R, Ehrenfels CW, Cate RL (1993) Detection of DNA in southern blots with chemiluminescence. Methods Enzymol 217:398–414. https://doi.org/10.1016/0076-6879(93)17079-k

    Article  CAS  PubMed  Google Scholar 

  17. Cartwright EJ (ed) (2009) Transgenesis techniques: principles and protocols editied by Elizabeth J. Cartwright, vol 561. Methods in molecular biology, 3rd edn. Springer/Humana Press, Totowa. https://doi.org/10.1007/978-1-60327-019-9

  18. Pluck A, Klasen C (2009) Generation of chimeras by morula aggregation. In: Cartwright EJ (ed) Transgenesis techniques. Methods in molecular biology, vol 561. Humana Press, New York, pp 219–229. https://doi.org/10.1007/978-1-60327-019-9_14

    Chapter  Google Scholar 

  19. Crawley JN, Belknap JK, Collins A, Crabbe JC, Frankel W, Henderson N, Hitzemann RJ, Maxson SC, Miner LL, Silva AJ, Wehner JM, Wynshaw-Boris A, Paylor R (1997) Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies. Psychopharmacology 132(2):107–124. https://doi.org/10.1007/s002130050327

    Article  CAS  PubMed  Google Scholar 

  20. Silva AJ, Simpson EM, Takahashi JS, Lipp HP, Nakanishi S, Wehner JM, Giese KP, Tully T, Chapman PF, Abel T, Fox K, Seth G, Itohara S, Lathe R, Mayford M, McNamara JO, Morris RJ, Picciotto M, Roder J, Shin HS, Slesinger PA, Storm DR, Stryker MP, Tonegawa S, Wang Y, Wolfer DP, Mice BCGB (1997) Mutant mice and neuroscience: recommendations concerning genetic background. Neuron 19(4):755–759. https://doi.org/10.1016/S0896-6273(00)80958-7

    Article  Google Scholar 

  21. Heffner CS, Herbert Pratt C, Babiuk RP, Sharma Y, Rockwood SF, Donahue LR, Eppig JT, Murray SA (2012) Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource. Nat Commun 3:1218. https://doi.org/10.1038/ncomms2186

    Article  CAS  PubMed  Google Scholar 

  22. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21(1):70–71. https://doi.org/10.1038/5007

    Article  CAS  PubMed  Google Scholar 

  23. Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13(1):133–140. https://doi.org/10.1038/nn.2467

    Article  CAS  PubMed  Google Scholar 

  24. Feldmeyer D, Kask K, Brusa R, Kornau HC, Kolhekar R, Rozov A, Burnashev N, Jensen V, Hvalby O, Sprengel R, Seeburg PH (1999) Neurological dysfunctions in mice expressing different levels of the Q/R site-unedited AMPAR subunit GluR-B. Nat Neurosci 2(1):57–64. https://doi.org/10.1038/4561

    Article  CAS  PubMed  Google Scholar 

  25. Bertocchi I, Eltokhi A, Rozov A, Chi VN, Jensen V, Bus T, Pawlak V, Serafino M, Sonntag H, Yang B, Burnashev N, Li SB, Obenhaus HA, Both M, Niewoehner B, Single FN, Briese M, Boerner T, Gass P, Rawlins JNP, Kohr G, Bannerman DM, Sprengel R (2021) Voltage-independent GluN2A-type NMDA receptor ca(2+) signaling promotes audiogenic seizures, attentional and cognitive deficits in mice. Commun Biol 4(1):59. https://doi.org/10.1038/s42003-020-01538-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Allen NM, Conroy J, Shahwan A, Lynch B, Correa RG, Pena SD, McCreary D, Magalhaes TR, Ennis S, Lynch SA, King MD (2016) Unexplained early onset epileptic encephalopathy: exome screening and phenotype expansion. Epilepsia 57(1):e12–e17. https://doi.org/10.1111/epi.13250

    Article  CAS  PubMed  Google Scholar 

  27. Endele S, Rosenberger G, Geider K, Popp B, Tamer C, Stefanova I, Milh M, Kortum F, Fritsch A, Pientka FK, Hellenbroich Y, Kalscheuer VM, Kohlhase J, Moog U, Rappold G, Rauch A, Ropers HH, von Spiczak S, Tonnies H, Villeneuve N, Villard L, Zabel B, Zenker M, Laube B, Reis A, Wieczorek D, Van Maldergem L, Kutsche K (2010) Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes. Nat Genet 42(11):1021–1026. https://doi.org/10.1038/ng.677

    Article  CAS  PubMed  Google Scholar 

  28. Bannerman DM, Bus T, Taylor A, Sanderson DJ, Schwarz I, Jensen V, Hvalby O, Rawlins JN, Seeburg PH, Sprengel R (2012) Dissecting spatial knowledge from spatial choice by hippocampal NMDA receptor deletion. Nat Neurosci 15(8):1153–1159. https://doi.org/10.1038/nn.3166

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bygrave AM, Masiulis S, Nicholson E, Berkemann M, Barkus C, Sprengel R, Harrison PJ, Kullmann DM, Bannerman DM, Katzel D (2016) Knockout of NMDA-receptors from parvalbumin interneurons sensitizes to schizophrenia-related deficits induced by MK-801. Transl Psychiatry 6(4):e778. https://doi.org/10.1038/tp.2016.44

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Hayashi Y, Nabeshima Y, Kobayashi K, Miyakawa T, Tanda K, Takao K, Suzuki H, Esumi E, Noguchi S, Matsuda Y, Sasaoka T, Noda T, Miyazaki J, Mishina M, Funabiki K, Nabeshima Y (2014) Enhanced stability of hippocampal place representation caused by reduced magnesium block of NMDA receptors in the dentate gyrus. Mol Brain 7(1):44. https://doi.org/10.1186/1756-6606-7-44

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kilonzo K, van der Veen B, Teutsch J, Schulz S, Kapanaiah SKT, Liss B, Katzel D (2021) Delayed-matching-to-position working memory in mice relies on NMDA-receptors in prefrontal pyramidal cells. Sci Rep 11(1):8788. https://doi.org/10.1038/s41598-021-88200-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Niewoehner B, Single FN, Hvalby O, Jensen V, Meyer zum Alten Borgloh S, Seeburg PH, Rawlins JN, Sprengel R, Bannerman DM (2007) Impaired spatial working memory but spared spatial reference memory following functional loss of NMDA receptors in the dentate gyrus. Eur J Neurosci 25(3):837–846. https://doi.org/10.1111/j.1460-9568.2007.05312.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Watanabe Y, Muller MK, von Engelhardt J, Sprengel R, Seeburg PH, Monyer H (2015) Age-dependent degeneration of mature dentate gyrus granule cells following NMDA receptor ablation. Front Mol Neurosci 8:87. https://doi.org/10.3389/fnmol.2015.00087

    Article  CAS  PubMed  Google Scholar 

  34. Hansen KB, Yi F, Perszyk RE, Furukawa H, Wollmuth LP, Gibb AJ, Traynelis SF (2018) Structure, function, and allosteric modulation of NMDA receptors. J Gen Physiol 150(8):1081–1105. https://doi.org/10.1085/jgp.201812032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Max Planck Institute for Medical Research, Heidelberg, Germany

    Rolf Sprengel

  2. Department of Biomedical Sciences, School of Medicine, Mercer University, Columbus, GA, USA

    Ahmed Eltokhi

  3. Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany

    Frank N. Single

Authors
  1. Rolf Sprengel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ahmed Eltokhi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Frank N. Single
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rolf Sprengel .

Editor information

Editors and Affiliations

  1. INSERM, Institut de Neurobiologie de la Méditerranée (INMED), Marseille, France

    Nail Burnashev

  2. INSERM, Institut de Neurobiologie de la Méditerranée (INMED), Marseille, France

    Pierre Szepetowski

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Sprengel, R., Eltokhi, A., Single, F.N. (2024). Generation of Rare Human NMDA Receptor Variants in Mice. In: Burnashev, N., Szepetowski, P. (eds) NMDA Receptors. Methods in Molecular Biology, vol 2799. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3830-9_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3830-9_6

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3829-3

  • Online ISBN: 978-1-0716-3830-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation