A modified method of nuclear transfer for investigating early development of mouse embryos reconstructed with cumulus cell nuclei

Xin ming Shen, Gui lin Qiao, Peizhou Jiang, Xin Li, Hua Huang, Kai tai Yao

Research output: Contribution to journalArticle

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Abstract

OBJECTIVE: To establish a fast, simple, efficient and minimally invasive method for nuclear transfer (NT) to study the early development of mouse embryos reconstructed with cumulus cell nuclei in vitro. METHODS: With a sharp-tipped enucleation needle, an incision approximately 25 mm in length was made in the zona pellucida of a rat oocyte, through which the first polar body was removed and the metaphase II chromosome-spindle complex was gently aspirated along with a minimal volume of the cytoplasm by slightly pressing and vacuum aspiration of the oocyte. A cumulus cell nuclei from C57BL/6j mouse, 10-12 mm in diameter, was inserted into the perivitelline space of the enucleated oocyte. The fusion of the donor-recipient pair was induced by electrofusion and nuclear formation was observed. The development of 2-cell, 4- to 8-cell and morula-stage embryos was observed after a 72-hour culture of the reconstructed oocytes in vitro. RESULTS: The modified NT method enabled one-step removal of the whole nucleus from the oocyte with confirmed reliability of complete nuclear removal by Hoechst 33342 staining of the removed nuclei examined under UV light. The process of enucleation took an average time of 15 s, and the survival rate of the enucleated oocytes reached 95%. The success rate of 76.7% was achieved for cumulus cell nucleus insertion into the zona pellucida of the enucleated oocytes and pronucleus formation occurred in 62.2% of the reconstructed oocytes with nuclear transfer. After 72 h of culture in vitro of the reconstructed oocytes in CZB medium, the percentage of embryos that developed into 2-cell, 4- to 8-cell and morula (more than 16 cells) stages were 57.5%, 39.1% and 27.6%, respectively. Microsatellite sequences (D7Mit22 and D4Mit204) were amplified from the DNA of the reconstructed embryos for identifying their origin, which was proved to be C57BL/6j mouse. CONCLUSION: The modified NT method is simple, minimally invasive, efficient and practicable to reconstruct mouse embryos with somatic cell nuclei.

Original languageEnglish (US)
Pages (from-to)613-618
Number of pages6
JournalDi 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA
Volume25
Issue number6
StatePublished - Jun 1 2005
Externally publishedYes

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Cumulus Cells
Cell Nucleus
Oocytes
Embryonic Development
Embryonic Structures
Morula
Zona Pellucida
Inbred C57BL Mouse
Polar Bodies
Oocyte Retrieval
Metaphase
Ultraviolet Rays
Vacuum
Microsatellite Repeats
Needles
Cytoplasm
Chromosomes
Staining and Labeling
DNA

ASJC Scopus subject areas

  • Medicine(all)

Cite this

A modified method of nuclear transfer for investigating early development of mouse embryos reconstructed with cumulus cell nuclei. / Shen, Xin ming; Qiao, Gui lin; Jiang, Peizhou; Li, Xin; Huang, Hua; Yao, Kai tai.

In: Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA, Vol. 25, No. 6, 01.06.2005, p. 613-618.

Research output: Contribution to journalArticle

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abstract = "OBJECTIVE: To establish a fast, simple, efficient and minimally invasive method for nuclear transfer (NT) to study the early development of mouse embryos reconstructed with cumulus cell nuclei in vitro. METHODS: With a sharp-tipped enucleation needle, an incision approximately 25 mm in length was made in the zona pellucida of a rat oocyte, through which the first polar body was removed and the metaphase II chromosome-spindle complex was gently aspirated along with a minimal volume of the cytoplasm by slightly pressing and vacuum aspiration of the oocyte. A cumulus cell nuclei from C57BL/6j mouse, 10-12 mm in diameter, was inserted into the perivitelline space of the enucleated oocyte. The fusion of the donor-recipient pair was induced by electrofusion and nuclear formation was observed. The development of 2-cell, 4- to 8-cell and morula-stage embryos was observed after a 72-hour culture of the reconstructed oocytes in vitro. RESULTS: The modified NT method enabled one-step removal of the whole nucleus from the oocyte with confirmed reliability of complete nuclear removal by Hoechst 33342 staining of the removed nuclei examined under UV light. The process of enucleation took an average time of 15 s, and the survival rate of the enucleated oocytes reached 95{\%}. The success rate of 76.7{\%} was achieved for cumulus cell nucleus insertion into the zona pellucida of the enucleated oocytes and pronucleus formation occurred in 62.2{\%} of the reconstructed oocytes with nuclear transfer. After 72 h of culture in vitro of the reconstructed oocytes in CZB medium, the percentage of embryos that developed into 2-cell, 4- to 8-cell and morula (more than 16 cells) stages were 57.5{\%}, 39.1{\%} and 27.6{\%}, respectively. Microsatellite sequences (D7Mit22 and D4Mit204) were amplified from the DNA of the reconstructed embryos for identifying their origin, which was proved to be C57BL/6j mouse. CONCLUSION: The modified NT method is simple, minimally invasive, efficient and practicable to reconstruct mouse embryos with somatic cell nuclei.",
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T1 - A modified method of nuclear transfer for investigating early development of mouse embryos reconstructed with cumulus cell nuclei

AU - Shen, Xin ming

AU - Qiao, Gui lin

AU - Jiang, Peizhou

AU - Li, Xin

AU - Huang, Hua

AU - Yao, Kai tai

PY - 2005/6/1

Y1 - 2005/6/1

N2 - OBJECTIVE: To establish a fast, simple, efficient and minimally invasive method for nuclear transfer (NT) to study the early development of mouse embryos reconstructed with cumulus cell nuclei in vitro. METHODS: With a sharp-tipped enucleation needle, an incision approximately 25 mm in length was made in the zona pellucida of a rat oocyte, through which the first polar body was removed and the metaphase II chromosome-spindle complex was gently aspirated along with a minimal volume of the cytoplasm by slightly pressing and vacuum aspiration of the oocyte. A cumulus cell nuclei from C57BL/6j mouse, 10-12 mm in diameter, was inserted into the perivitelline space of the enucleated oocyte. The fusion of the donor-recipient pair was induced by electrofusion and nuclear formation was observed. The development of 2-cell, 4- to 8-cell and morula-stage embryos was observed after a 72-hour culture of the reconstructed oocytes in vitro. RESULTS: The modified NT method enabled one-step removal of the whole nucleus from the oocyte with confirmed reliability of complete nuclear removal by Hoechst 33342 staining of the removed nuclei examined under UV light. The process of enucleation took an average time of 15 s, and the survival rate of the enucleated oocytes reached 95%. The success rate of 76.7% was achieved for cumulus cell nucleus insertion into the zona pellucida of the enucleated oocytes and pronucleus formation occurred in 62.2% of the reconstructed oocytes with nuclear transfer. After 72 h of culture in vitro of the reconstructed oocytes in CZB medium, the percentage of embryos that developed into 2-cell, 4- to 8-cell and morula (more than 16 cells) stages were 57.5%, 39.1% and 27.6%, respectively. Microsatellite sequences (D7Mit22 and D4Mit204) were amplified from the DNA of the reconstructed embryos for identifying their origin, which was proved to be C57BL/6j mouse. CONCLUSION: The modified NT method is simple, minimally invasive, efficient and practicable to reconstruct mouse embryos with somatic cell nuclei.

AB - OBJECTIVE: To establish a fast, simple, efficient and minimally invasive method for nuclear transfer (NT) to study the early development of mouse embryos reconstructed with cumulus cell nuclei in vitro. METHODS: With a sharp-tipped enucleation needle, an incision approximately 25 mm in length was made in the zona pellucida of a rat oocyte, through which the first polar body was removed and the metaphase II chromosome-spindle complex was gently aspirated along with a minimal volume of the cytoplasm by slightly pressing and vacuum aspiration of the oocyte. A cumulus cell nuclei from C57BL/6j mouse, 10-12 mm in diameter, was inserted into the perivitelline space of the enucleated oocyte. The fusion of the donor-recipient pair was induced by electrofusion and nuclear formation was observed. The development of 2-cell, 4- to 8-cell and morula-stage embryos was observed after a 72-hour culture of the reconstructed oocytes in vitro. RESULTS: The modified NT method enabled one-step removal of the whole nucleus from the oocyte with confirmed reliability of complete nuclear removal by Hoechst 33342 staining of the removed nuclei examined under UV light. The process of enucleation took an average time of 15 s, and the survival rate of the enucleated oocytes reached 95%. The success rate of 76.7% was achieved for cumulus cell nucleus insertion into the zona pellucida of the enucleated oocytes and pronucleus formation occurred in 62.2% of the reconstructed oocytes with nuclear transfer. After 72 h of culture in vitro of the reconstructed oocytes in CZB medium, the percentage of embryos that developed into 2-cell, 4- to 8-cell and morula (more than 16 cells) stages were 57.5%, 39.1% and 27.6%, respectively. Microsatellite sequences (D7Mit22 and D4Mit204) were amplified from the DNA of the reconstructed embryos for identifying their origin, which was proved to be C57BL/6j mouse. CONCLUSION: The modified NT method is simple, minimally invasive, efficient and practicable to reconstruct mouse embryos with somatic cell nuclei.

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