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Mouse Genome Engineering Using Designer Nucleases
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Genomic DNA damage in mouse transgenesis.

Yasuhiro Yamauchi1, Brendan Doe, Anna Ajduk

  • 1Institute for Biogenesis Research, University of Hawaii Medical School, Honolulu, Hawaii 96822, USA.

Biology of Reproduction
|July 27, 2007
PubMed
Summary

Creating transgenic mammals is inefficient due to chromosomal damage. Both pronuclear microinjection and intracytoplasmic sperm injection (ICSI) cause DNA damage, impacting embryo development and transgenic success rates.

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Area of Science:

  • Reproductive Biology
  • Genetics
  • Developmental Biology

Background:

  • Transgenic mammal creation is inefficient, with high embryo loss.
  • Existing methods face challenges in gene integration and expression.
  • Embryo loss is linked to chromosomal abnormalities during development.

Purpose of the Study:

  • To investigate the causes of embryo loss in transgenic mammal production.
  • To compare chromosomal integrity and developmental outcomes across different transgenesis techniques.
  • To identify the mechanisms of DNA damage in various assisted reproductive technologies.

Main Methods:

  • Examined chromosome integrity in embryos produced via pronuclear microinjection, ICSI, and IVF-mediated transgenesis.
  • Assessed embryo development in vitro and transgenic morula/blastocyst yield.
  • Utilized comet assay to detect DNA damage in sperm prior to ICSI.

Main Results:

  • Pronuclear microinjection (both transgenic and sham) caused significant oocyte arrest and chromosome breaks, independent of transgenes.
  • ICSI showed lower chromosomal damage than pronuclear injection and was dependent on exogenous DNA.
  • Sperm DNA damage was detected prior to ICSI, correlating with observed chromosomal damage.
  • IVF-mediated gene transfer did not impact chromosome integrity but yielded no transgenic embryos.
  • ICSI resulted in better embryonic development and transgene expression compared to pronuclear microinjection.

Conclusions:

  • DNA damage is a significant factor in the inefficiency of transgenic mammal creation.
  • Both pronuclear microinjection and ICSI induce DNA damage through distinct mechanisms.
  • ICSI appears more promising for improving embryo development and transgenic efficiency despite associated DNA damage concerns.