Transgenic Mouse Production by Microinjection of Oocytes
The Investigator provides purified DNA suitable for microinjection experiments, appropriate documentation about fragment preparation, and documentation of an established protocol for identifying transgenic founder animals carrying the transgene.
If at all possible, the DNA fragment to be injected must be separated from plasmid -vector components by restriction digest, followed by agarose gel electrophoresis and recovery of the fragment from the gel. There are a variety of procedures that are acceptable for this purpose, including the QIAEX II™ gel extraction kit or Mo Bio UltraClean™ GelSpin® Kit, electroelution, glass-milk adsorption, and recovery by agarase treatment, which are described in Manipulating the mouse embryo: Hogan, B. et al.; Cold Spring Harbor Laboratory Press. 2nd edition, pages 228-232 (see protocols). Only reagents of highest available quality should be used. The final elution of the fragment should be performed with injection buffer [10 mM Tris-HCl, pH 7.4; 0.1 mM EDTA] prepared with embryo-certified water (SIGMA). The Investigator provides the purified fragment in injection buffer at a concentration of > 30 micrograms/ml . The amount and purity of the purified fragment is determined by UV absorption (spectrum from 220 to 300 nm), and by comparative gel analysis. The latter is achieved by running an aliquot of the fragment on an agarose gel side by side with various known amounts of the parental plasmid digested to release the fragment to be analyzed. The concentration of the isolated fragment can be judged by comparing the relative intensities of the bands after ethidium bromide staining. Documentation about fragment preparation and analysis (functional vector map, size, relevant restriction sites, UV spectrum, Gel analysis) is submitted to the core with the fragment.
Documentation of a genotyping protocol for identifying transgenic founder mice by southern blot analysis or by PCR-analysis of genomic DNA must be provided by the investigator before initiation of injection experiments. Functional assays for detection of the transgene are not sufficient, and not required.
The Core guarantees at least two transgenic founders or 30 pups per injection experiment. Genomic DNA will be prepared from tail biopsies of 3-4 week old offspring by proteinase K digestion/phenol extraction/ethanol precipitation. If no transgenic founders are obtained after the 30 pups are screened, the Core will discuss with the Investigator how to further proceed. We do not guarantee expression of the transgene. A founder is defined as any mouse with either the full or partial DNA construct.
The Core routinely uses oocytes from B6D2F1 hybrid females, mated to C57Bl/6J males for injection experiments, yielding transgenic founders on a 75% C57Bl/6 genetic background. By request, the Core also can generate transgenic founders on a C57Bl/6J or NOD background. Fees for the generation of transgenic founders from this line, for injection of non-standard DNA constructs, such as BACs, or other non-standard procedures may vary.
To initiate an experiment, contact the TG Core Supervisor and submit a completed order form for zygote microinjections. Injections will be performed at the earliest possible time, usually beginning within 4 weeks after receipt of the DNA construct. Offspring will be born 3 weeks after injection, and DNA will be made available 4 weeks thereafter. Fee information is available from the TG Core Supervisor.