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Related Experiment Videos

Effect of microinjection time during postfertilization S-phase on bovine embryonic development

M Gagné1, F Pothier, M A Sirard

  • 1Unité de recherche en Ontogénie et Reproduction, Centre Hospitalier de l'Université Laval, Québec, Canada.

Molecular Reproduction and Development
|June 1, 1995
PubMed
Summary

Timing microinjection during bovine zygote DNA replication is crucial for embryo development and gene expression. Mid-S phase injection (18 hpi) yielded significantly better results for both embryo development and marker gene expression.

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Area of Science:

  • Reproductive Biology
  • Developmental Biology
  • Molecular Biology

Background:

  • Bovine zygote development and gene expression are sensitive to manipulation during early stages.
  • DNA replication timing is a critical window for successful genetic modification in zygotes.
  • Understanding optimal microinjection timing is essential for improving in vitro embryo production.

Purpose of the Study:

  • To determine the optimal timing for microinjection during bovine zygote DNA replication.
  • To evaluate the impact of microinjection timing on subsequent in vitro embryo development.
  • To assess the expression of an injected gene construct based on microinjection timing.

Main Methods:

  • Bovine zygotes were cultured in vitro and DNA replication was assessed using 3H-thymidine incorporation.

Related Experiment Videos

  • Aphidicolin and cycloheximide were used to synchronize zygotes and arrest/resynchronize DNA replication and protein synthesis.
  • Microinjection of a chicken beta-actin promoter-lac Z gene construct was performed at early (15 hpi), mid (18 hpi), and late (21 hpi) S phase.

    • Main Results:

    • Embryonic development was significantly higher in zygotes injected at mid-S phase (18 hpi) compared to early or late S phase.
    • Marker gene expression was observed in later developmental stages (>16 cells) only in zygotes injected at 18 hpi.
    • Gene expression levels correlated with developmental stage, being highest when injection occurred during mid-zygotic S phase.

    Conclusions:

    • Microinjection timing during the S phase of bovine zygotes critically influences both embryo development and transgene expression.
    • Optimal microinjection for improved embryo survival and gene expression occurs at approximately 18 hours post-insemination.
    • Cytoplasmic processing, rather than pronuclear processing, appears to be the primary factor affected by microinjection timing.