Services and Pricing

Pronuclear DNA Microinjection

Pronuclear DNA microinjection has been the most popular method of producing genetically altered mice. This service is initiated when an investigator fills out a request form that includes a description of the DNA fragment to be injected, provides an approved animal care protocol number, billing and grant information, and a brief description of the DNA to be injected. After approval by a member of our staff, the DNA fragment provided is fluorometrically-quantitated, filtered, and is normally diluted to 3 ng/ul for microinjection. Three to five days of microinjection experiments are then performed in order to provide the investigator with at least 20 pups. Provided that the injected construct integrates into the mouse chromosomal DNA at the one-cell stage, the animal will contain the injected DNA. If integration of the injected DNA construct occurs at a later stage of embryo development, the resulting animal will be mosaic.

DNA is microinjected into the male pronucleus of a 0.5 day mouse embryo. After transfer of injected embryos to the oviduct of a pseudopregnant recipient, the animals are housed under the protocol number of the investigator. The gestation period of the mouse is 19 to 21 days. Once pups are born, they will be ready for weaning at 3 to 4 weeks of age.

The investigator will be notified of potential transgenic pups as weaning of these pups nears. Pups will be weaned, tailed and numbered by core personnel at weaning. The investigator will need to perform DNA isolation and analyze the DNA by PCR and Southern blotting.

Two or more of the pups are usually transgenic if the investigator has provided a DNA fragment of adequate quality. If no transgenic models are generated, a decision will need to be made after reviewing potential reasons for the low efficiency as to whether reinjection will occur, if a re-prep of the DNA to be injected is needed, or if analysis of potential transgenic embryos is deemed necessary due to the possibility of the DNA construct resulting in embryonic lethality.

Several factors influence the production of transgenic mice. Of utmost importance for successful outcome is the quality and size of the DNA to be injected, thus no guarantee is made by the Transgenic Mouse/Embryonic Stem Cell Shared Resource for the number of transgenic mice produced. However, provided that the DNA has been prepared in the recommended manner, 10 to 20% of the pups born will usually carry the transgene. The facility has generated over 1,400 founder animals since the initiation of the shared resource. We will make every effort to make transgenic mice with the DNA provided, but will not guarantee success with every construct.