Genotyping Genetically Modified (GM) Mice

  • Neeraj K. AryalEmail author
  • Jan Parker-ThornburgEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2066)


Prior to generating a new mouse model, it is important to plan the method that will be used to detect which of the mice generated have the mutation(s) desired. Nearly, all types of mutations may be detected using PCR. However, the choice of primers will differ depending upon the method used to generate the model. Transgenic mice should be genotyped across a unique junction fragment. Targeted ES cells used to generate knock-out or knock-in mice should be genotyped using primers from a unique marker in the construct and a region outside of the construct. Targeting in ES cells can also be detected using a genomic Southern blot. Mice targeted using CRISPR/Cas9 should have the region of interest amplified using PCR, and then be assessed for size changes (for large changes in sequence) by Surveyor Assay (for gene knock-out and point mutations) and/or sequenced to verify the mutation. Each of these models has a unique requirement for genotyping, and failure to understand the requirements can easily lead to loss of the gene in subsequent generations.

Key words

Genotyping Genetically engineered mice Transgenic Targeted Knock-out mice Knock-in mice CRISPR/Cas9 Germ line transmission Chimera Mosaic PCR 



The authors would like to thank Dr. Vinod Pant for sharing his expertise with Southern blot analysis. This work was supported by a Cancer Center Support Grant NCI # CA016672(GEMF).


  1. 1.
    Singh P, Schimenti JC, Bolcun-Filas E (2015) A mouse geneticist’s practical guide to CRISPR applications. Genetics 199:1–15. Scholar
  2. 2.
    Yen ST, Zhang M, Deng JM, Usman SJ, Smith CN, Parker-Thornburg J, Swinton PG, Martin JF, Behringer RR (2014) Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev Biol 393:3–9. Scholar
  3. 3.
    Behringer R (ed) (2014) Manipulating the mouse embryo: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  4. 4.
    Molto E, Vicente-Garcia C, Montoliu L (2011) Designing transgenes for optimal expression. In: Pease S, Saunders TL (eds) Advanced protocols for animal transgenesis. Springer, New YorkGoogle Scholar
  5. 5.
    Nagy A, Gertsenstein M, Vintersten K, Behringer RR (2004) Detection and analysis of mouse genome alterations and specific sequences. In: Manipulating the mouse embryo, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  6. 6.
    Saunders TL (2011) Gene targeting vector design for embryonic stem cell modifications. In: Pease S, Saunders TL (eds) Advanced protocols for animal Transgenesis. Springer, New YorkGoogle Scholar
  7. 7.
    Brennan K (2011) Colony management. In: Pease S, Saunders TL (eds) Advanced protocols for animal transgenesis. Springer, New YorkGoogle Scholar
  8. 8.
    Hnatyszyn HJ, Podack ER, Young AK, Seivright R, Spruill G, Kraus G (2001) The use of real-time PCR and fluorogenic probes for rapid and accurate genotyping of newborn mice. Mol Cell Probes 15:169–175. Scholar
  9. 9.
    Thomsen N, Ali RG, Ahmed JN, Arkell RM (2012) High resolution melt analysis (HRMA); a viable alternative to agarose gel electrophoresis for mouse genotyping. PLoS One 7:e45252. Scholar
  10. 10.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. Scholar
  11. 11.
    Harms DW, Quadros RM, Seruggia D, Ohtsuka M, Takahashi G, Montoliu L, Gurumurthy CB (2014) Mouse genome editing using the CRISPR/Cas system. Curr Protoc Hum Genet 83(15.7):11–27. Scholar
  12. 12.
    Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE (2016) Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 34:339–344. Scholar
  13. 13.
    Lauer M (2016) Authentication of key biological and/or chemical resources in NIH grant applications.
  14. 14.
    Yang B, Wen X, Kodali NS, Oleykowski CA, Miller CG, Kulinski J, Besack D, Yeung JA, Kowalski D, Yeung AT (2000) Purification, cloning, and characterization of the CEL I nuclease. Biochemistry 39:3533–3541CrossRefGoogle Scholar
  15. 15.
    Brown T (2001) Hybridization analysis of DNA blots. Curr Protoc Immunol. Chapter 10: Unit 10.16B.

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Genes and Development Program, Department of Genetics, Graduate School for Biomedical SciencesMD Anderson Cancer CenterHoustonUSA
  2. 2.Bioscience, Oncology R&DAstraZenecaBostonUSA
  3. 3.Department of Genetics, Genetically Engineered Mouse FacilityMD Anderson Cancer CenterHoustonUSA

Personalised recommendations