Transgenics - Pronuclear microinjection

Service Description

The centers will microinject DNA into pronuclei of fertilized mouse oocytes. The investigator must provide a DNA construct of high quality, prepared using one of the protocols approved by the service centers. After injection the center will transfer the embryos to pseudo pregnant females. When the pups are two - three weeks old they will be ear tagged and tail DNA will be purified and sent to the investigator for transgene detection, which should take 2-3 weeks. Once the founders have been identified, the investigator will be billed and the Center will wean the mice and transfer them to the investigator.

This procedure will generate transgenic mice which have one or more copies of the transgene integrated into a random site of the genome. A minimum of three transgenic mice are guaranteed for each DNA construct using C57Bl6XC3H strains.

Mice maintained in the center and not transferred to the researcher, after they have been analyzed and confirmed to be transgenic by PCR and or Southern will result in the billing of additional housing (cage) charges to the researcher.

Responsibility of the Investigator

• Provide the DNA and ensure it is prepared according to our specifications (DNA prep.). The purity of the DNA is important to ensure there are no contaminants (agarose, EtBr, ethanol, phenol, salts etc) which will harm the embryo. There are a number of proven commercial kits available to purify DNA (Table 7.1 Manipulating the mouse embryo, A Laboratory Manual, Nagy et Al., Cold Spring Harbor Laboratory Press, p304.).
• 2 ug of the transgene in TE (low salt) or H₂O at a concentration of 50ng/ul or higher.
• A minigel photo of a small quantity of the DNA to ensure it is intact, the correct size and not denatured.
• A PCR assay that demonstrates that the transgene can be detected at a single copy when mixed with mouse tail DNA (provided by the Center).
• Analysis of the tail samples provided by the investigator (some centers can perform the testing at an additional cost).
• Maintenance charges when mice are in our care 2 weeks past the time that tail samples were available.
• Download the request form Here and e-mail to (M. Chagnon) or fax to (514) 398-6769.

Guarantees
A minimum of 250-300 zygotes are injected for each DNA construct. The mice are housed under specific pathogen free conditions. We guarantee the production of at least 3 transgene positive pups. We can not guarantee that mice positive for the transgene will result in the desired expression of the protein nor even that the transgene will be transmitted to the F1 generation.

Personnel Training / Support

• A recommended protocol for microinjection DNA purification is available
• Assistance in establishing methods of transgene detection
• Training in the microinjection of DNA and production of genetically altered mice (only at certain centers, please send inquires to M. Chagnon)