Faites glisser Méthode de préparation pour préserver tridimensionnelle de la chromatine architecture des cellules germinales du testicule

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Namekawa, S. H. Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells. J. Vis. Exp. (83), e50819, doi:10.3791/50819 (2014).

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Abstract

Materials

Name Company Catalog Number Comments
Cot-1 DNA  Invitrogen 18440-016
Stratagene Prime-It Fluor Fluorescence Labeling Kit Agilent 300380
Herring sperm DNA Solution Invitrogen 15634-017
Ethanol. 200 proof (absolute) Pharmco-aaper 111000200
Sodium acetate Sigma-Aldrich S2889
Formamide deionized American Bioanalytical AB00600-00500
Sodium citrate tribasic dihydrate Sigma-Aldrich C8532
Bovine serum albumin Lifeblood Medical 77110-100
Dextran sulfate sodium salt from Leuconostoc spp. Sigma-Aldrich D6001
RPMI 1640 Invitrogen A10491-01
Sucrose Sigma-Aldrich S0389
PIPES Sigma-Aldrich P6757
Magnesium chloride hexahydrate Sigma-Aldrich M0250
Triton X-100 Sigma-Aldrich T8787
Paraformaldehyde Sigma-Aldrich 76240
Sodium hydroxide Sigma-Aldrich 221465
Phosphate buffered saline Sigma-Aldrich P4417
VECTASHIELD Mounting Medium with DAPI Vector Laboratories H1200
Ribonucleoside-vanadyl complex New England Biolabs S1402S
Illustra MicroSpin G-50 Columns GE Healthcare 27-5330-01
Microcentrifuge Eppendorf 5424
Forceps VWR 300-050
Microcentrifuge tubes Bioexpress C-3262-1
Cytospin 3 Shandon
Coplin Jar Fisher 08-815
Superfrost /Plus Microscope Slides Fisher 12-550-15
4-well dish Fisher 12-566-301
Cover glass  Fisher 12-544-G
Air incubator Revolutionary Science RS-IF-203

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References

  1. Peters, A. H., Plug, A. W., van Vugt, M. J., de Boer, P. A drying-down technique for the spreading of mammalian meiocytes from the male and female germline. Chromosome Res. 5, 66-68 (1997).
  2. Namekawa, S. H., et al. Postmeiotic sex chromatin in the male germline of mice. Curr Biol. 16, 660-667 (2006).
  3. Namekawa, S. H., Lee, J. T. Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos. Nat Protoc. 6, 270-284 (2011).
  4. Mahadevaiah, S. K., Costa, Y., Turner, J. M. Using RNA FISH to study gene expression during mammalian meiosis. Methods in molecular biology. 558, 433-444 (2009).
  5. Hall, L. L., et al. An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells. Proc Natl Acad Sci U S A. 99, 8677-8682 (2002).
  6. Huynh, K. D., Lee, J. T. Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos. Nature. 426, 857-862 (2003).
  7. Namekawa, S. H., VandeBerg, J. L., McCarrey, J. R., Lee, J. T. Sex chromosome silencing in the marsupial male germ line. Proceedings of the National Academy of Sciences of the United States of America. 104, 9730-9735 (2007).
  8. Namekawa, S. H., Payer, B., Huynh, K. D., Jaenisch, R., Lee, J. T. Two-step imprinted X inactivation: repeat versus genic silencing in the mouse. Mol Cell Biol. 30, 3187-3205 (2010).
  9. Ichijima, Y., et al. MDC1 directs chromosome-wide silencing of the sex chromosomes in male germ cells. Genes Dev. 25, 959-971 (2011).
  10. Sin, H. S., et al. RNF8 regulates active epigenetic modifications and escape gene activation from inactive sex chromosomes in post-meiotic spermatids. Genes Dev. 26, 2737-2748 (2012).

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