Transgenic mice are most commonly produced by microinjection of DNA into the pronuclei of fertilized single-cell mouse embryos. Transgene integration is random, with multiple copies of the transgene typically integrating into a single chromosomal locus in the embryo. If integration takes place prior to the first nuclear division, then all cells will carry the transgene. After injection, the eggs are surgically transferred to the oviducts of time-mated pseudopregnant foster mothers, generated by mating females with vasectomized males. The offspring resulting from injected eggs may or may not carry the transgene. On average, about 10-20% of the mice born will test positive for the transgene. The mice that do carry the transgene are called founders.
- purchase and housing of mice used to produce embryos
- superovulation of egg donors and mating with stud males
- creation of pseudopregnant foster mothers by mating outbred ICR females with vasectomized males and checking for vaginal plugs the morning of injection
- harvesting of eggs from euthanized donors
- dilution of the client’s transgene DNA with injection buffer to a concentration of 2 ng/ul and injection of this solution into one pronucleus of each fertilized egg
- surgically transfer all eggs that survive the injection process into the oviducts of pseudopregnant foster mothers
- monitoring of foster mothers before and after birth of pups
- weaning, ear-tagging, and tail-tipping pups
- provide tail biopsies to the client for genotyping
- arrange and assist with the transfer or shipment of transgenic mice to the investigator’s mouse colony
The classic transgene design includes an enhancer and promoter, the mRNA coding sequence and a complete set of poly-adenylation signals. A splice donor, intron and splice acceptor between the coding sequence and the poly-adenylation signals often increases expression. The construct should be designed so that the transgene insert can be purified away from plasmid backbone by gel purification.
Transgene Construct Preparations
The investigator should provide 30 µl or more of transgene DNA fragment at a minimum concentration of 50 ng/µl in filtered microinjection buffer (10 mM Tris, pH 7.4; 0.25 mM EDTA).
- Using restriction enzymes, remove as much of the vector sequence as possible (the plasmid may interfere with expression of your transgene).
- Many commercial kits are available for plasmid and insert purification: Qiagen Endo-Free Plasmid Maxi Kit, QIAquick PCR Purification Kit, and the UltraClean GelSpin Kit.
- Use sterile endotoxin-free, ultra-pure water from Sigma (Sigma W1503) to make injection buffer. Injection buffer can also be purchased from Millipore (MR-095-10F).
- Resuspend recovered DNA in filtered (0.22 µm) microinjection buffer.
- Run the DNA on a minigel and make sure that it is intact, of right size, and there is no smear of sheared DNA.
- The cleanliness of the construct is of utmost importance. It should be absolutely free of any phenol, chloroform, ethidium bromide, high salt and ethanol. Even trace amounts will quickly lyse the injected embryos or interfere with the development of the implanted embryos.
The standard background strain used for the embryos is B6C3 (C57BL/6 x C3H). Embryos are produced by mating B6C3 females to C57BL/6 males, generating potential founder animals which are 75% C57BL/6 and 25% C3H. Other strains can be used as egg donors, but these will require additional fees. Some strains do not respond to superovulating hormones and/or have poor quality embryos and should be avoided if possible.
Prices and Guarantees
- We guarantee that at least 70 injected embryos will be implanted into psuedo pregnant recipients, or 2 founders produced, whichever comes first. The construct DNA provided by the investigator must be clean and non-toxic.
- In the event that an alternate host embryo strain is required, the additional costs incurred from purchasing and housing a special strain of stud males, will be charged to the investigator. The investigator will also pay the difference in purchase price between the cost of B6C3 females typically used, and the cost of the embryo host strain selected. No guarantee will be made as to the number of injected embryos transferred, due to strain variability in embryo production.
- C57BL/6 embryos are available at no additional cost per injection day, but without guarantee. This strain is less efficient. Thus three injection sessions are typically required to achieve the same results as one session of B6C3 injection.
- The investigator pays per diem cage charges beginning on the day of embryo transfer.
- Cancellation of injection within two weeks of the scheduled date will result in a $250 fee to cover the cost of the embryo donor mice purchased for the injection.
- Click here for current rates.
Reserve An Injection Session
To order an ES cell injection session, go to our TRP Forms page. Fill out a “Request for Pronuclear Microinjection” form and e-mail it to Bob Hunter email@example.com. Be sure to include a current budget number and your IACUC animal use protocol number.
Constructs may be delivered to the Health Sciences Building, room T-140, during normal business hours. Ask for Serina Tsang firstname.lastname@example.org or Bob Hunter email@example.com.
International Society of Transgenic Technologie