Unfertilized Embryos  

 
            Two Cell Embryo

             
           Multiple Cell Embryos

The LARC MMPI service microinject DNA is for investigators in UCSF who have generated transgenic DNA construct in their lab. Investigator provided purified DNA is microinjected into the male pronuclear of single-cell fertilized mouse embryos (zygotes). Microinjected embryos are surgically transferred into the reproductive tract of recipient females. Once pups are weaned, LARC MMPI service will initiate the paperwork for the transfer of these mice to investigor’s animal room and protocol.

Transgenic DNA integrates randomly into the embryonic genome following microinjection. Usually, multiple copies of the transgene integrate at a single chromosomal site just prior to the first cell division, which ensures that the transgene is present in all nucleated cells of the developing embryo. Approximately 10-30% of the microinjected embryos that develop to term will inherit the transgene, or 2-7 expected founders for most constructs. LARC MMPI makes no guarantee that transgenic founders will occur.

LARC will not issue refunds for injection services that do not generate mutant mice

Time-line (about 10 weeks):

• Week 1: Microinjection are scheduled and mice are ordered
• Week 2: mice arrival; preparation of donor and recipient females
• Week 3: DNA received, microinjection and surgical transfer of injected embryos
• Week 6: pups born 19 days following surgery
• Week 9-10: pups weaned and transferred to PI’s barrier room

The LARC MMPI service microinjects ES cells for investigators in UCSF who have generated targeted clones in their lab. Investigator provided ES cells are microinjected into the cavity of an expanded blastocyst stage embryo and injected embryos are surgically transferred into the reproductive tract of recipient females. Once chimeras are weaned, LARC MMPI service will initiate the paperwork for the transfer of these mice to your animal room and protocol.

Not all ES cell clones will result in the production of chimeric mice and not all chimeric mice are capable of transmitting the mutated allele though the germline. The ability to generate germline transmitting chimeras depends on the pluripotent capacity of the microinjected ES cells. Keeping ES cells in a non-differentiated state is largely a function of how carefully the lab generating handles the cells during culture.

We cannot guarantee that microinjections will result in the generation of chimeric mice or that chimeric animals that are produced will be germline competent, as there is no way of knowing how cells were grown or handled by others prior to arriving in the lab.

LARC will not issue refunds for injection services that do not generate mutant mice.

Timeline for scheduled blastocyst injections( about 10 weeks):

• Week 1: ordering of embryo donor mice
• Week 2: mice arrival; preparation of donor and recipient females
• Week 3: receiving of ES cells and blastocyst microinjection
• Week 6: pups born 17 days following blastocysts microinjection
• Week 10: 3-4 week old chimeric pups weaned, and transferred to PI.

TOTAL COSTS:

•  We estimate the cost to be $1600, however the actual charge depends on the hours expended on the injection process. (Assumes 200 embryo injection for DNA injection, and 100 blastocysts for ES injection).
• Investigator must provide Found/DPA for LARC to set-up an LARC recharging code to buy embryo donor mic, CD-1 females as pseudo pregnant recipients and B6D2 males as Vasectamized males at an additional recharge.
• The mice housing and transfer fee will be charged to PI’s LARC research  billing code. Technical services are charged at the current hourly rate for injection services at $61.00 per hour.

PLACE AN ORDER:

Investigators must have an IACUC approved animal protocol describing the use of animals generated by the LARC MMPI service before microinjections can be scheduled. Send a completed Request Form to [email protected].

 - Information and materials needed:

    DNA microinjection:

• · Linear maps of transgenic construct

  · Photo of microinjection DNA gel showing a sharp, single transgenic fragment band of the correct size

• Purified microinjection quality DNA

• For plasmid DNA:

• • 5 to 10 µg of linear high quality (ultrapure) microinjection DNA diluted in buffer supplied by TE buffer at a concentration about100µg/ml

• For BAC DNA:

• • High quality, fresh BAC DNA analyzed by pulse-field gel electrophoresis for purity and concentration, diluted in buffer supplied by TE at a concentration of ~50µg/ml

    ES microinjection:

• Investigators must prepare cells for microinjection in ES growth medium

• Bring the prepared cells on ice to the laboratory by 11:00 am on the scheduled injection day.

For questions please contact: [email protected]

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