The submission of every DNA prep should be accompanied by:
a) a proof that the transgene can be detected in genomic DNA by a functional assay (PCR or Southern blot).
b) a gel photo where integrity and quantity of the DNA is documented with the help of a known DNA marker and a non-digested DNA sample.
A total amount of at least 5 μgr DNA, is required.
All documentation sent to the Facility should be provided electronically. If the DNA prep meets all requirements we will ask the investigator to proceed with the shipment.
Female donor mice are superovulated and mated with fertile males. Zygotes are recovered and injected with the diluted DNA.
We use Elutip column to purify plasmid DNA, in order to make sure it will be microinjection-grade clean. In the case of injection of big DNA fragments, there is available a protocol for preparation of BAC DNA upon request.
Injected zygotes are transferred into foster mothers. Following approximately 10 days after pups are born, we take tail biopsies and we perform a standard phenol protocol for DNA extraction provided to the investigators for genotype screening. If a different protocol is required for extraction, according to the screening assay to be used, then the investigator receives the tail biopsies.
The results of genotype screening and transgenic founder identification should be obtained ideally by weaning time, the latest when the mice are four weeks old. For extended periods of the animal maintenance additional costs might apply for external users.
The minimum of 3 transgenic founders will be delivered for every construct injected.
DNA microinjections are routinely performed on B6;CBA (F2) zygotes. Alternatively, C57Bl6 zygotes can be used, as well as other strains after discussion.
In collaboration with Fleming’s Flow Cytometry facility (www.fleming.gr/en/services/flow/index.html) our facility may provide analysis of the identified founders to confirm the expression of the inserted transgene.