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Analysis of Recombinants in Female Mouse Meiosis

  • Esther de Boer
  • Maria Jasin
  • Scott KeeneyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 957)

Abstract

During meiosis, homologous chromosomes (homologs) undergo recombinational interactions, resulting in the formation of crossovers (COs) or noncrossovers (NCOs). Both COs and NCOs are initiated by the same event: programmed double-strand DNA breaks (DSBs), which occur preferentially at hotspots throughout the genome. COs contribute to the genetic diversity of gametes and are needed to promote proper meiotic chromosome segregation. Accordingly, their formation is tightly controlled. In the mouse, the sites of preferred CO formation differ between male and female chromosomes, both on a regional level and on the level of individual hotspots. Sperm typing using (half-sided) allele-specific PCR has proven a powerful technique to characterize COs and all detectable NCOs at hotspots on male human and mouse chromosomes. In contrast, very little is known about the properties of hotspots in female meiosis. This chapter describes an adaptation of sperm typing to analyze recombinants in a hotspot, using DNA isolated from an ovary cell suspension enriched for oocytes.

Key words

Meiosis Recombination Crossover Noncrossovers Hotspot Allele-specific PCR Oocyte 

Notes

Acknowledgements

We thank Liisa Kauppi and Francesca Cole for advice on allele-specific PCR. This work was supported by a Netherlands Organization for Scientific Research Rubicon Grant 825.07.006 (E.B.) and a National Institutes of Health Grant R01 HD53855 (S.K. and M.J.).

References

  1. 1.
    Keeney S (2007) Spo11 and the formation of DNA double-strand breaks in meiosis. In: Lankenau DH (ed) Recombination and meiosis. Springer, Heidelberg, Germany, pp 81–123Google Scholar
  2. 2.
    Allers T, Lichten M (2001) Differential timing and control of noncrossover and crossover recombination during meiosis. Cell 106:47–57PubMedCrossRefGoogle Scholar
  3. 3.
    Hunter N, Kleckner N (2001) The single-end invasion: an asymmetric intermediate at the double-strand break to double-holliday junction transition of meiotic recombination. Cell 106:59–70PubMedCrossRefGoogle Scholar
  4. 4.
    Kauppi L, Jeffreys AJ, Keeney S (2004) Where the crossovers are: recombination distributions in mammals. Nat Rev Genet 5:413–424PubMedCrossRefGoogle Scholar
  5. 5.
    Jeffreys AJ, Kauppi L, Neumann R (2001) Intensely punctate meiotic recombination in the class II region of the major histocompatibility complex. Nat Genet 29:217–222PubMedCrossRefGoogle Scholar
  6. 6.
    Guillon H, de Massy B (2002) An initiation site for meiotic crossing-over and gene conversion in the mouse. Nat Genet 32:296–299PubMedCrossRefGoogle Scholar
  7. 7.
    Jeffreys AJ, May CA (2004) Intense and highly localized gene conversion activity in human meiotic crossover hot spots. Nat Genet 36:151–156PubMedCrossRefGoogle Scholar
  8. 8.
    Guillon H, Baudat F, Grey C, Liskay RM, de Massy B (2005) Crossover and noncrossover pathways in mouse meiosis. Mol Cell 20:563–573PubMedCrossRefGoogle Scholar
  9. 9.
    Holloway K, Lawson VE, Jeffreys AJ (2006) Allelic recombination and de novo deletions in sperm in the human beta-globin gene region. Hum Mol Genet 15:1099–1111PubMedCrossRefGoogle Scholar
  10. 10.
    Cole F, Keeney S, Jasin M (2010) Comprehensive, fine-scale dissection of homologous recombination outcomes at a hot spot in mouse meiosis. Mol Cell 39:700–710PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Paigen K et al (2008) The recombinational anatomy of a mouse chromosome. PLoS Genet 4(7):e1000119PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Billings T et al (2010) Patterns of recombination activity on mouse chromosome 11 revealed by high resolution mapping. PLoS ONE 5(12):e15340PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    de Boer E et al (2006) Two levels of interference in mouse meiotic recombination. Proc Natl Acad Sci U S A 103:9607–9612PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Bois PR (2007) A highly polymorphic meiotic recombination mouse hot spot exhibits incomplete repair. Mol Cell Biol 27:7053–7062PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Smagulova F et al (2011) Genome-wide analysis reveals novel molecular features of mouse recombination hotspots. Nature 472:375–378PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Jeffreys AJ, Murray J, Neumann R (1998) High-resolution mapping of crossovers in human sperm defines a minisatellite-associated recombination hotspot. Mol Cell 2:267–273PubMedCrossRefGoogle Scholar
  17. 17.
    Hubert R et al (1994) High resolution localization of recombination hot spots using sperm typing. Nat Genet 7:420–424PubMedCrossRefGoogle Scholar
  18. 18.
    Kauppi L, May CA, Jeffreys AJ (2009) Analysis of meiotic recombination products from human sperm. Methods Mol Biol 557:323–355PubMedCrossRefGoogle Scholar
  19. 19.
    Cole F, Jasin M (2011) Isolation of meiotic recombinants from mouse sperm. Methods Mol Biol 745:251–282PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Baudat F, de Massy B (2009) Parallel detection of crossovers and non-crossovers in mouse germ cells. Methods Mol Biol 557:305–322PubMedCrossRefGoogle Scholar
  21. 21.
    Eppig JJ, Schroeder AC (1989) Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro. Biol Reprod 41:268–276PubMedCrossRefGoogle Scholar
  22. 22.
    McClellan KA, Gosden R, Taketo T (2003) Continuous loss of oocytes throughout meiotic prophase in the normal mouse ovary. Dev Biol 258:334–348PubMedCrossRefGoogle Scholar
  23. 23.
    Wood WI et al (1985) Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci USA 82:1585–1588PubMedCrossRefGoogle Scholar
  24. 24.
    Jeffreys AJ, Neumann R (2002) Reciprocal crossover asymmetry and meiotic drive in a human recombination hot spot. Nat Genet 31:267–271PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Institut de Génétique et MicrobiologieUniversité Paris-SudOrsayFrance
  2. 2.Developmental Biology ProgramMemorial Sloan-Kettering Cancer CenterNew YorkUSA
  3. 3.Molecular Biology ProgramMemorial Sloan-Kettering Cancer CenterNew YorkUSA
  4. 4.Howard Hughes Medical InstituteNew YorkUSA

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