In aggregation chimeras between diploid embryos and tetraploid embryos, tetrapoloid cells are found only in extra-embryonic tissues, the trophoblast of the placenta and the endoderm layer of the yolk sac. The typical use of these chimeras in phenotypic analysis is in instances when gene-manipulated mice have an extra-embryonic phenotype. When diploid embryos are derived from mutant mice and tetraploid embryos from wild-type mice, wild-type tetraploid embryos can contribute to the defective trophoblast and primitive endoderm derivatives of mutant mice. When embryonic lethality of mutant mice is rescued in the aggregation chimeras between mutant diploid embryos and wild-type tetraploid embryos, the embryonic lethality is caused by the defect in extra-embryonic tissues, not embryonic tissues. |
Category | Glycogene transgenic animals |
Protocol Name | Tetraploid rescue experiment |
Authors
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Asano, Masahide
Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University
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Reagents
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KSOM-AA medium for embryo culture (Merck Millipore, Billerica, MA, Cat. #MR-121-D) |
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M2 medium for embryo manipulation (Sigma-Aldrich, St. Louis, MO, Cat. #M7167) |
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0.3 M Mannitol (Sigma-Aldrich, Cat. #M4125) |
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Mineral oil for embryo culture (Sigma-Aldrich, Cat. #M8410) |
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Acidic Tyrode’s solution (Sigma-Aldrich, Cat. #T1788) |
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Instruments
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Pulse generator (CF-150B, BLS Ltd., Budapest, Hungary) |
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Electrode-chamber with 500-μm gap (GGS-500, BLS Ltd.) |
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60-mm and 100-mm petri dish |
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Mouth pipet for embryo manipulation |
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Aggregation needle (DN-09, BLS Ltd.) |
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Methods |
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Production of tetraploid embryos (1)
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Two-cell stage embryos are collected in M2 medium from superovulated female mice mated with male mice. If CAG-EGFP Tg mice are used, tetraploid cells are marked with green fluorescence. |
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A 100-mm petri dish containing the electrode chamber (GSS-500) is set under a dissecting microscope. |
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Two large drops of M2 medium (drops 1 and 4) and two drops of 0.3 M mannitol/0.3% BSA solution (drops 2 and 3) are placed in the dish. Drop 3 should be eccentric with respect to the electrodes. |
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The two-cell stage embryos are placed in the M2 drop 1. |
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About 30 embryos are rinsed through the mannitol drop 2 and placed in the mannitol drop 3 between the electrodes. |
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The embryos are oriented parallel to each other by the AC field (2V). |
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A short electric pulse (50 V for 35 msec, twice) is applied to the embryos with the pulse generator (CF-150B). |
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The embryos are transferred into the M2 drop 4. |
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Steps 5–8 are repeated for the remaining embryos, taking no longer than 15 min. |
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The embryos are washed through several drops of KSOM-AA medium and placed into the KSOM-AA drops under mineral oil in CO2 incubator (37°C, 5% CO2). |
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The embryos are then fused to produce one-cell-stage tetraploid embryos. |
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At 30–45 min later, the complete fused tetraploid embryos are separated to new KSOM-AA drops. |
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The fused tetraploid embryos are cultured in KSOM-AA medium for 24 h to develop to the 4-cell-stage tetraploid embryos, which are used for aggregation with diploid embryos. |
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Aggregation chimeras between diploid embryos and tetraploid embryos (1)
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Aggregation plates are prepared as follows. About 20 deep depressions are created in a 60-mm petri dish using the aggregation needle (DN-09) and the depressions are covered by 20 μL of the KSOM-AA drops and mineral oil. |
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Eight-cell-stage embryos are collected from superovulated female mice mated with male mice. |
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Zona pellucidae are removed from the 8-cell-stage diploid embryos and the 4-cell-stage tetraploid embryos by acidic Tyrode’s solution. |
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Zona-free diploid embryos and tetraploid embryos are aggregated in aggregation plates. |
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The aggregated embryos are cultured in KSOM-AA medium overnight to make morulae or blastocysts. |
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The chimeric embryos are then transferred to the uterus of pseudo pregnant ICR females. |
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Chimeric embryos are collected at the intended days after transplantation or developed to birth. |
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The presence of the EGFP gene in embryonic and extra-embryonic tissues is examined by fluorescence and PCR. |
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Discussion | We applied the tetraploid rescue experiment to clarify the lethal cause of β4GalT-5-/- embryos (2). β4GalT-5-/- embryos aggregated with wild-type tetraploid embryos developed to at least E12.5, demonstrating that the embryonic lethality of the β4GalT-5-/- embryos that occurred by E10.5 was caused by defects in extra-embryonic tissues (Fig. 1). This idea is supported by the observations that β4GalT-5-/- embryos exhibited hematoma and accumulated trophoblast giant cells abnormally in the anti-mesometrial pole of the extra-embryonic tissues (2). However, β4GalT-5-/- embryos aggregated with wild-type tetraploid embryos did not develop to E18.5 (Table 1). This finding suggests that β4GalT-5 is essential for the embryonic tissues during late embryogenesis as well as for extra-embryonic tissues during early embryogenesis. |
Figure & Legends |
Figure & Legends
Fig. 1. β4GalT-5-/- embryos rescued by wild-type tetraploid embryos.
(A, B) Embryos were dissected at E9.5 after being aggregated with wild-type tetraploid embryos. (C, D) Sections of the embryo and extra-embryonic tissue in B were stained with HE. (E, F) Embryos were dissected at E12.5 after being aggregated with wild-type tetraploid embryos. A, E: β4GalT-5+/- embryos, B, C, D, F: β4GalT-5-/- embryos. (G) PCR genotyping of the β4GalT-5 allele (left) and EGFP transgene (right) in embryos and ectoplacental cones dissected at E9.5. Note that the No.5 and No.8 β4GalT-5-/- embryos were rescued by wild-type tetraploid embryos. mt, β4GalT-5 mutant band; wt, β4GalT-5 wild-type band. Scale bars, 200 μm in C and D.
This figure was originally published in Glycobiology 20: 1311–1322, 2010 “β4-Galactosyltransferase-5 is a lactosylceramide synthase essential for mouse extra-embryonic development” Nishie et al. Oxford University Press.
Table 1
This table was originally published in Glycobiology 20: 1311–1322, 2010 “β4-Galactosyltransferase-5 is a lactosylceramide synthase essential for mouse extra-embryonic development” Nishie et al. Oxford University Press. |
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Date of registration:2014-12-24 14:22:48 |
- Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. (2003) Production of Chimeras. Manipulating the Mouse Embryo: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY pp453–506.
- Nishie, T., Hikimochi, Y., Zama, K., Fukusumi, Y., Ito, M., Yokoyama, H., Naruse, C., Ito, M., and Asano, M. (2010) β4-Galactosyltransferase-5 is a lactosylceramide synthase essential for mouse extra-embryonic development. Glycobiology 20, 1311–1322 [PMID : 20574042]
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