Transgenic Mouse Service Descriptions

CRISPR Injection

New clients consult with Jun Zhao
Client designs and generates single guide RNA (sg RNA) and ss oligo/ ds fragment if applicable.
Core can provide client with Cas 9 mRNA (acquired from Life Technologies) for a nominal fee.
Client prepares RNA injection mixture to give to the Core for injection.
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.

Pronuclear Injection

A desired DNA construct is injected into the pronucleus of a 0.5 d.p.c. embryo. Resulting pups are returned to the client. The client will then analyze these by southern blot /PCR to determine the presence of the given transgene. Clients will report the results of screen to core.
Read more here.

Blastocyst Injection

Mouse embryonic stem cells 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.

Commercially purchased ES cell Maintenance

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

Gene Targeting

A targeting vector containing a 'knock-out' or 'knock-in' gene of interest is electroporated into mouse embryonic stem cells (129 SvJ or C57 Bl6). The cells undergo drug selection, clones are picked and expanded in tissue culture, then returned to the client for analysis. Homologous recombinant clones are verified by the client using either PCR or southern blot analysis by the client. The additional cost for C57Bl6 targeting is for an exclusive medium “RESGRO” that the core uses to grow these ES cells in.
Read more here.

RMCE Electroporation

RMCE (recombinase mediated cassette exchange) enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After "gene swapping" the donor cassette is safely locked in but can nevertheless be re-mobilized in case other compatible donor cassettes are provided ("serial RMCE").

Chromosome Count

Chromosome counting is done on targeted homologous recombinant ES cell clones prior to blastocyst injection for quality control.

Embryo Re-Derivation

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

Ovary Transplants

Sexually mature females that are infertile or cannot carry a litter to term are ovariectomized. These ovaries are then transplanted into the wild-type ovariectomized females of a given background strain, and immediately returned to the client. Two weeks later, the recipients are mated by the client to produce transgenic pups of the original genotype.

Embryo Freezing

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

In- vitro Fertilization (IVF)

Using IVF, we can routinely produce dozens of siblings that can be used to jump start a breeding colony or perform characterization studies from even strains with poor breeding efficiency. We need one healthy male to start with or we are able to resuscitate the line using frozen sperm.

Client provides at least two vials/straws of sperm for the line to be generated, or one male for fresh sperm
Core acquires wild-type females of appropriate age and strain
Core performs IVF
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

Sperm Freezing

Up to four mouse lines will be frozen on a given day. $303.00 for first line plus $100.00 for every additional line. (A 10 percent discount is available for cancer center members.) 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
Core will freeze sperm
Eighteen vials are frozen per line
Core will store vials for up to one week. Client does long-term storage in liquid N2

Embryo Thawing

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

For service and rates table please link individual service to corresponding description/bullet points.

Tailing

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.