Transgene detection during early murine embryonic development after pronuclear microinjection

Area of Science:

• Reproductive Biology
• Developmental Biology
• Molecular Biology

Background:

• Pronuclear microinjection is a common technique for generating transgenic animals.
• Assessing the impact of microinjection on early embryonic development is crucial for optimizing transgenic strategies.

Purpose of the Study:

• To evaluate the effects of pronuclear microinjection of a whey acidic protein (WAP) gene transgene on mouse embryo development.
• To determine the efficiency of transgene detection in embryos at various developmental stages and in arrested/fragmented embryos.

Main Methods:

• Polymerase chain reaction (PCR) was used to detect the endogenous WAP gene and the WAP-human protein C transgene in mouse ova.
• Mouse ova were cultured in vitro to different developmental stages (one-cell, two-cell, four-cell, morula, blastocyst) after pronuclear microinjection.
• Developmental rates and embryo quality were assessed after microinjection with DNA construct, buffer alone, or no injection.

Main Results:

• The WAP transgene was detected in a high percentage of early-stage embryos, but detection decreased in later stages.
• Embryos microinjected with the DNA construct showed significantly reduced development to the blastocyst stage compared to controls.
• High concentrations of the injected DNA construct were associated with lower developmental rates and higher rates of embryo fragmentation and arrest.

Conclusions:

• Pronuclear microinjection of the WAP transgene negatively impacts mouse embryonic development.
• The microinjection procedure itself, or the presence of the transgene construct, can lead to developmental arrest and fragmentation.
• Optimizing DNA construct concentration and injection procedures is necessary to improve developmental outcomes in transgenic mouse production.