Pronuclear Microinjection

Please note, this service is intended for labs with extensive molecular biology, gene targeting, and genotyping expertise. We do offer a full-service option in collaboration with the ISCRM Ellison Stem Cell Core. Please inquire.

• purchase and housing of mice used to produce embryos for injection
• superovulation of embryo 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 embryos from euthanized donors
• dilution of the client’s transgene DNA with injection buffer and/or prepare RNP
• microinjection of investigator’s gRNA/Cas9/DNA repair template into 1-cell stage mouse embryos
• surgically transfer all embryos 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 investigator is responsible for designing, testing, and preparing the transgene construct/gRNA/repair 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. "Sticky" DNA or RNA preps will clog the injection needles and kill embryos. Great care should be taken when preparing the injection mix as the success of the procedure is highly dependent upon the quality of the reagents  being injected into the embryos. See Protocols page for more information.

• The investigator is responsible for genotyping all pups.
• The investigator should establish a PCR protocol that can reliably detect your transgene/mutation at the single copy level when mixed with wild-type mouse DNA. This should be completed before the construct is injected into mouse embryos.

The standard background strain used for the embryos is B6D2 (C57BL/6 x DBA/2J). Embryos are produced by mating B6D2 females to C57BL/6N males, generating potential founder animals which are 75% C57BL/6N and 25% DBA/2J. Other strains can be used as egg donors, but these may require additional fees. Some strains do not respond to superovulating hormones and/or have poor quality embryos and should be avoided if possible. Pure C57BL/6N embryos may be requested, but survival and efficiency are generally poor compared to hybrid strains.

• We guarantee that at least 200 injected embryos will be surgically transferred into pseudopregnant recipients, or 2 founders produced, whichever comes first. The reagents 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 standard hybrid strains, 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/6NJ embryos are available at no additional cost. This strain tends to be less efficient compared to hybrid strains.
• Click here for current rates.

To schedule a Pronuclear Microinjection session, go to our TRP Forms page. Fill out a “Request for Pronuclear Microinjection” form and e-mail it to Robert J Hunter. Please be sure to acknowledge the Transgenic Resources Program (RRID:SCR_017863) in your publications.