Submission of DNA
For transgenes less than 20kb in size, the UNC Animal Models Core Facility generally creates transgenic mice from DNA provided by the client. Transgene cloning and purification services are available upon request. BAC transgene DNAs are always prepared by the Core for injection. For an optimum number of founders, the transgene plasmid should be purified using an endotoxin-free system, such as the Qiagen EndoFree system (catalog #12362). All or most of the plasmid backbone should be cut from the transgene DNA with restriction enzymes in such a way that the transgene DNA is easily separated from the plasmid backbone by agarose gel electrophoresis. The client should supply the facility with 20 µg of the digested DNA. The client must also supply primers to be used for genotyping potential founders, and demonstrate that the assay can detect their transgene by PCR when mixed with genomic DNA at an appropriate ratio to mimic 1 transgene copy per genome.
Clients requesting BAC transgenic services should contact the core director for procedures for submitting BAC clones to the core. The core's BAC transgenic pricing includes BAC DNA preparation and pulsed-field gel electrophoresis.
All clients are required to complete the "AMC Request Form" before project work can be initiated. The transgene DNA will be injected into the pronuclei of C57BL/6 or FVB embryos and implanted into pseudo-pregnant recipient females. Pups are born approximately 3 weeks after the injections and genotyping is provided by the core. Founders will be transferred to the client as soon as animals are weaned. The facility's pronuclear microinjection service includes injection of up to 200 embryos or production of 3 founders, whichever is achieved first. The goal of the facility is to generate a minimum of 3 transgenic founder animals for each experiment. Different founder lines may display distinct expression levels, some founders may not transmit the transgene to their offspring, and some lines may not express the transgene at all. The production of 3 founders gives a reasonable chance to obtain at least 1 good transgenic line. The facility makes no guarantees with respect to founder production, germline transmission or gene expression in the founders and their offspring. A deleterious transgene may result in an inability to obtain founders or to obtain germline-transmission or expression in the offspring. If no transgenic animals are obtained after injection of 200 embryos, the client will be billed for the service and a consultation with the facility director is advised before attempting additional microinjection projects with that transgene. Animal space at UNC must be requested from DLAM and must be secured prior to project initiation. Failure to secure animal space will result in additional charges and could result in the loss of valuable animals.
For a detailed description of the protocols involved in generating a transgenic mouse see: Ittner LM, Götz J. Pronuclear injection for the production of transgenic mice. Nat Protoc. 2007;2(5):1206-15.