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Resources and Recommendations
Standard plasmid transgenes: The transgene DNA produced by the customer can be sent as either intact supercoiled plasmid DNA or as purified injection-ready transgene insert. To obtain high quality DNA the purification of the plasmid for microinjection can be done by centrifugation in a CsCl gradient. Other purification options also exist as many laboratories are successful using DNA purification kits as simple and fast tools to achieve microinjection quality DNA.
Preparation of DNA for Pronuclear Injection
1. Purification of supercoiled plasmids by one of these methods:
a) GenElute Plasmid Miniprep Kit (Sigma Cat. No NA0150).
b) UltraClean™GelSpin™Kit (Mo Bio Laboratories, Cat. No 12400-250).
c) Centrifugation in a CsCl gradient.
2. Cut the injection fragment out of the cloning vector using the appropriate restriction enzymes. Final yield should be 10 - 20 micrograms of transgene fragment.
3. Separate restriction digest products on an agarose gel using either TBE or TAE buffer. Use either low- or standard- melting temperature agarose. Make sure the gel is run long and slow enough to isolate the fragment from the vector.
4. Place gel on a transilluminator. Use a clean razor blade or scalpel to cut out the transgene fragment. Minimize the time of exposure to UV-light.
5. Use the Qiaquick gel extraction kit from Qiagen (Cat. No. 28704). Do not use Vortex if your fragment is larger than 10 kb.
6. Quantitate DNA solution.
7. Verify size the intact condition of the DNA on a minigel.
8. Adjust the concentration of the purified transgene to 50 - 100 ng/µl and submit frozen DNA sample containing at least 0.5 µg of DNA to TMM.
9. Transgenic Core will dilute, aliquot and store the eluted DNA at -200 C.
BAC transgenes: BAC transgenes can be injected into zygotes of one of our standard strains of mice as indicated below;either in the inbred C57BL/6 strain or in the F1-hybrid strain (B6D2F1). For BAC transgenic mice please purify the DNA by CsCl centrifugation, and send as linearized DNA at a concentration of approximately 50 –100 ng/µl. Prior to injecting the BAC transgene, we will dilute and equilibrate the DNA in a buffer containing salt and polyamines.
Inbred C57BL/6 transgenic mice: Currently majority of our customers request transgenic mice on the C57BL/6 inbred background because the genome and phenotype of this mouse line is well known. This background is also advantageous if the transgenic line is crossed with induced mutants that are maintained as congenic strains on this background. We offer the production of transgenic mice on the C57BL/6 inbred background with either plasmid or BAC transgenes. However, because the overall efficiency is lower in C57BL/6, we charge higher fees than for the F1-hybrid transgenic mice.
F1-hybrid mouse strain: The C57BL/6 x DBA/2 F1 hybrid strain has been used in this core for standard transgene microinjections. Under the terms of the agreement, we will produce at least 30 offspring from the injections of pronuclear stage embryos. These animals will be transferred to the PI's colony at 3 weeks of age (weaning age).
Responsible Parties and Timeline
Before initiating a transgenic mouse project please receive approval of the Institute Animal Care and Use Committee (IACUC) and the Institute Biosafety Committee (IBC) for your animal use and recombinant DNA protocols. Then fill out, sign and submit the Project Request Form to TMM.
In addition to the Project Request Form, please provide the following materials:
1) A map of the construct and a simplified description of the phenotype you expect to produce.
2) A restriction digest containing approximately 2-4 micrograms of your plasmid.
3) A photo of an aliquot of the digest analyzed by agarose gel electrophoresis with the transgene band clearly identified.
Service fees include:
a) Purchase of virgin C57BL6/J or (B6xDBA-2)F1 females (donors of oocytes).
b) Superovulation of the virgin females.
c) Microinjection into 250-300 C57BL/6 or B6xDBA-2 zygotes.
d) Transplantation of the microinjected embryos into pseudopregnant recipient females.
e) TMM guarantees the production of 3 transgenic founders or 40 total pups, whichever comes first. TMM does not guarantee expression of the transgene.
f) Animal housing charges for recipients and their offspring until weaning.
Service fees do not include:
a) Animal housing charges for litters after weaning.
b) Ear/tail tissue collection for DNA isolation for genotyping.
c) Health testing of the foster mother.
d) Shipping charges.
e) Customers will be charged per diem while mice remaining within the Transgenic Core's mouse room after weaning.
According to the customers' request, TMM can purchase and use other mouse lines for the generation of genetically modified mice. Moreover, TMM can tag, collect tail samples, extract DNA and genotype the offspring by PCR. For this purpose the animals can be housed in our colony beyond the usual 3 weeks (weaning age). These additional services will be charged as extra fees including the extra costs for the cage(s) per diem.
See Fees, Transgene Microinjections.