Abstract
An increasing interest in the molecular mechanisms governing cell division has resulted in the discovery of several groups of genes that participate in the regulation of mitosis and meiosis in eukaryotes. Inactivation of these genes in mice often leads to early embryonic lethality. To show direct causality between mutations of these genes, chromosomal instability and embryonic death, a technique enabling detailed cytogenetic analysis of embryonic cells is required. Here, we develop and test a comprehensive approach that allows complex analysis of individual early postimplantation embryos and combines polymerase chain reaction genotyping with the preparation and detailed karyotypic inspection of cells at the metaphase and anaphase stages. The method enables good chromosomal spreading and scattering of nuclei to perform routine cytogenetics (i.e., standard stain and G-banding). It also permits the application of specialized techniques such as fluorescence in situ hybridization to detect particular chromosomes and to verify the integrity of individual chromosomes. Utility of the new method is demonstrated by an analysis of embryonic day E7.5–E9.5 tissue from mice deficient in the spindle checkpoint gene Bub1b.
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Abbreviations
- DAPI:
-
Diamidophenylindole
- DMEM:
-
Dulbecco’s modified eagle’s medium
- FISH:
-
Fluorescence in situ hybridization
- MEFs:
-
Mouse embryonic fibroblasts
- MSC:
-
Mitotic spindle checkpoint
- PCR:
-
Polymerase chain reaction
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Acknowledgments
We gratefully acknowledge Michael Schmid for providing lab space, reagents, Y chromosome specific probe and advice. We are grateful to Claus Steinlein and Galina Fedorova for expert technical assistance. We thank Burkhard Kneitz for the gift of Bub1b knockout mice and stimulation of helpful discussion, Pamela Stanley for WW-6 ES cells, Elaine Huang for critical reading of this manuscript, and Malcolm Low for discussions and editing of the final text. This work was partly supported by the IZKF of the University of Wuerzburg, Germany and by Medical Research Foundation of Oregon, USA (grant GRESE0010A).
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Tian, Q., Hanlon Newell, A.E., Wang, Y. et al. Complex cytogenetic analysis of early lethality mouse embryos. Chromosome Res 19, 567–574 (2011). https://doi.org/10.1007/s10577-011-9209-4
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DOI: https://doi.org/10.1007/s10577-011-9209-4