Service Details

• New clients consult with Sang Lee at srl001@health.ucsd.edu. 
• Core can provide CRISPR design if desired, contact Sang Lee.
• Client generates single guide RNA (sg RNA) and ss oligo or ds fragment if applicable.
• Client prepares RNA injection mixture to give to the Core for injection. Core can provide guidelines for the preparation of the mixture, contact Sang Lee.
• Core prepares pronuclear stage embryos.
• Core injects RNA mix into the embryos.
• Core implants embryos into foster females.
• Core gets ACP to transfer 7- to 10-day-old pups and foster moms to client’s vivarium.
• Client screens resulting pups for transgene.
• For strains other than C57BL/6NHsd, i.e., Special Order, client is responsible for the cost of the males.

Mouse embryonic stem cells (ES) that have undergone homologous recombination are injected into the blastocoel cavity of 3.5 d.p.c. blastocyst stage C57Bl6C57Bl6 albino embryos. Injected blastocysts are implanted into pseudopregnant recipient females, and chimeric pups are born. Chimerics are returned to the Client. Those that transmit the 'knock-out' allele to germline produce agouti/black pups, which are analyzed for phenotype by the client.

• Core will thaw and prep ES cell clones for scheduled blastocyst injection
• Core injects es cells into either C57Bl6 blastocysts or albino C57BL6 blastocysts as required by strain of es cell
• 35-40 blastocysts injected per clone implanted into 5-6 recipient Dams
• Chimeras born get transferred to client at day 10.

This service includes thawing, expanding, freezing away cell stocks, mycoplasma test and chromosome counts. The core will also prep the cells for a blastocyst injection when required. The price of $523.00 includes special media requirements per cell line and mycoplamsa test and chromosome count for the first clone. Each additional clone will cost $200.00 more.

• Client provides protocol for ES cell growth and culture conditions purchased to core
• Client co-ordinates purchased ES cells drop off to core
• Core will grow cells, freeze vials of clone and give back pellet to client for reconfirmation, do a chromosome count and mycoplasma test

Embryo Rederivation is used to create a pathogen free strain of mice from lines that arrive at the university from a non-approved vendor and mice contaminated with various pathogens (MHV, Pinworm, Parvovirus). Males arrive in the rodent isolation unit (RIU), and wild-type females of the desired background strain are superovulated and mated to the males. Embryos are harvested in the RIU, washed and transferred into pathogen free, pseudopregnant recipient females by the core, pups are transferred to client after clean bill of health.

• Client coordinates the receipt of at least two males ages between 2 to 5 months, of desired strain into the RIU with the Animal Care Program Acquisitions Office.
• Core acquires wild-type females of the appropriate background strain and schedules procedure
• Core super-ovulates and RIU personnel mate females with males in RIU
• Core collects embryos, washes them and transfers into the oviducts of pseudo-pregnant recipient females
• Recipient females are retained in CMM-E quarantine until new pups are weaned.
• Pups are weaned and the dams are tested for pathogens
• Once dams are deemed clean, then pups are transferred to destination vivarium
• Client genotypes the transferred pups

Genetic banking through the use of mouse embryo cryopreservation is a practical means to store scientifically valuable mice. Mouse embryos stored in liquid nitrogen offer a safe way to preserve lines with potential future use but no current use and offer savings in facility space and the expense associated with keeping a breeding colony. Another advantage is that during the process of embryo collection, most pathogenic organisms are excluded as in other methods used to rederive lines of mice.

Briefly, the procedure entails harvesting embryos from donor females at the 8-cell developmental stage that is optimal for cryopreservation by our method. A controlled-rate methanol bath freezer is used to gradually cool the embryos to -80°C, before plunging them into liquid nitrogen for storage. Quality control is assured by freezing and thawing a representative sample of wild-type frozen embryos along with each line frozen down. Frozen embryos should be retrieved by the investigator one week post-freezing for long-term storage in liquid nitrogen in the investigator's own lab.

• Client coordinates the receipt of at least 8 to 9 males preferably homozyogous for transgene, ages between 2 to 5 months, of desired strain into the Rodent Isolation Unit (RIU) with the Animal Care Program Acquisitions Office.
• Core acquires wild-type females of the appropriate background strain and schedules procedure
• Core super-ovulates and RIU personnel mate females with males in RIU
• Core collects embryos, and freezes them.
• Core will freeze up to 200 embryos per line (if homozygous males provided) and 250 embryos per line ( if heterozygous males provided)
• Core will store vials for up to 1 week. Client does long-term storage in liquid N2

Up to eight mouse lines can be frozen on a given day. Recharge rate shown is for the first line. An additional $100.00 is recharged for each additional line frozen on the same day. The core would like two proven breeder males at the appropriate age (12 weeks to 20 weeks of age) per line.

• Client will send two males at 3 to 5 months of age (proven breeders) to the RIU for each line to be frozen, or the Core personnel can come to the Client’s lab if the mice are brought to the lab from their vivarium.
• Core will freeze sperm.
• Eighteen vials are frozen per line.
• Client does long-term storage in liquid N2.

The Core is able to resuscitate a mouse line using frozen embryos provided by investigators.

We have routinely thawed frozen embryos using protocols accompanying the embryos, and implanted all recovered embryos to produce pups.

• Client provides at least two vials/straws of frozen embryos to be regenerated
• Core will thaw and implant embryos into recipient females
• Recipient females are retained in CMM-E quarantine until pups are weaned
• Pups are weaned and dam is tested for pathogens
• Pups are transferred to destination vivarium
• Client genotypes the transferred pups

Service Rates

The Core will tail litters and send these back to investigators for screening.

This is not a routine service we offer, but done only when non-UCSD labs are not able to make mouse transfers in time. The investigators will get charged the per diems for cage costs and tailing costs.