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Tetraploid rescue experiment
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Tetraploid rescue experiment

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Introduction Protocol References Credit lines
Category
Glycogene transgenic animals
Protocol Name

Tetraploid rescue experiment

Authors
Asano, Masahide
Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University
KeyWords
Reagents

KSOM-AA medium for embryo culture (Merck Millipore, Billerica, MA, Cat. #MR-121-D)

M2 medium for embryo manipulation (Sigma-Aldrich, St. Louis, MO, Cat. #M7167)

0.3 M Mannitol (Sigma-Aldrich, Cat. #M4125)

Mineral oil for embryo culture (Sigma-Aldrich, Cat. #M8410)

Acidic Tyrode’s solution (Sigma-Aldrich, Cat. #T1788)

Instruments

Pulse generator (CF-150B, BLS Ltd., Budapest, Hungary)

Electrode-chamber with 500-μm gap (GGS-500, BLS Ltd.)

Dissecting microscope

CO2 incubator

60-mm and 100-mm petri dish

Mouth pipet for embryo manipulation

Aggregation needle (DN-09, BLS Ltd.)

Methods
1.

Production of tetraploid embryos (1)

1) 

 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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2) 

 A 100-mm petri dish containing the electrode chamber (GSS-500) is set under a dissecting microscope.

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3) 

 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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4) 

 The two-cell stage embryos are placed in the M2 drop 1.

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5) 

 About 30 embryos are rinsed through the mannitol drop 2 and placed in the mannitol drop 3 between the electrodes.

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6) 

 The embryos are oriented parallel to each other by the AC field (2V).

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7) 

 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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8) 

 The embryos are transferred into the M2 drop 4.

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9) 

 Steps 5–8 are repeated for the remaining embryos, taking no longer than 15 min.

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10) 

 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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11) 

 The embryos are then fused to produce one-cell-stage tetraploid embryos.

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12) 

 At 30–45 min later, the complete fused tetraploid embryos are separated to new KSOM-AA drops.

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13) 

 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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2.

Aggregation chimeras between diploid embryos and tetraploid embryos (1)

1) 

 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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2) 

 Eight-cell-stage embryos are collected from superovulated female mice mated with male mice.

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3) 

 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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4) 

 Zona-free diploid embryos and tetraploid embryos are aggregated in aggregation plates.

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5) 

 The aggregated embryos are cultured in KSOM-AA medium overnight to make morulae or blastocysts.

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6) 

 The chimeric embryos are then transferred to the uterus of pseudo pregnant ICR females.

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7) 

 Chimeric embryos are collected at the intended days after transplantation or developed to birth.

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8) 

 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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