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Transgenic cloned mice expressing enhanced green fluorescent protein generated by activation stimuli combined with

C-H Chen1, L Stone, J-C Ju

  • 1Division of Biotechnology, Animal Technology Institute Taiwan, Chunan, Miaoli, Taiwan.

Reproduction in Domestic Animals = Zuchthygiene
|March 4, 2008
PubMed

Insights

This study demonstrates that 6-dimethylaminopurine (6-DMAP) combined with strontium and cytochalasin B effectively activates reconstructed mouse oocytes for somatic cell nuclear transfer (SCNT). This novel activation method resulted in successful full-term development of cloned mice.

Area of Science:

  • Reproductive Biology
  • Developmental Biology
  • Genetics

Background:

  • Traditional mouse cloning relies on strontium (Sr) and cytochalasin B (CB) for oocyte activation.
  • 6-dimethylaminopurine (6-DMAP), a protein kinase inhibitor, has not been successfully applied in mouse cloning.
  • Investigating novel activation methods is crucial for improving SCNT efficiency.

Purpose of the Study:

  • To evaluate the activation efficiency of 6-dimethylaminopurine (6-DMAP) in mouse somatic cell nuclear transfer (SCNT).
  • To compare different activation treatments and culture media for cloned embryo development.
  • To generate cloned mice using 6-DMAP-based activation protocols.

Main Methods:

  • Oocytes were reconstructed using somatic cell nuclear transfer (SCNT) with donor cells from EGFP-expressing ESC chimera F1.
  • Activation treatments included combinations of strontium (Sr), 6-dimethylaminopurine (6-DMAP), and cytochalasin B (CB).
  • Cloned embryos were cultured in KSOM-AA, MEM-alpha, or MK media, and development rates were assessed.

Main Results:

  • The Sr6D (Sr + 6-DMAP) and Sr6D + SrCB treatments yielded higher parthenogenetic blastocyst rates (71-72%).
  • The Sr6D + SrCB group showed significantly higher hatching and hatched blastocyst rates (31%).
  • MEM-alpha medium supported better morula/blastocyst (40%) and blastocyst (29%) development rates.
  • Four enhanced green fluorescent protein (EGFP) cloned mice were successfully generated using the 6-DMAP activation protocol.

Conclusions:

  • 6-dimethylaminopurine (6-DMAP) in combination with Sr and CB effectively activates reconstructed mouse oocytes for SCNT.
  • The developed protocol supports the full-term development of transgenic SCNT cloned embryos.
  • This study establishes a new, efficient activation method for mouse cloning.

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