Genetic Models Core

CRISPR-Cas9 can generate multiple indel mutations in a single mouse. This service will characterize the spectrum of indel alleles all mutants produced by pro-nuclear injection. PCR products from the modified locus will be amplified, cloned, and sequenced. The sequences will be aligned with the wild type allele and possible frame-shift and pre-mature stop codons identified.

Core personnel will design and test sgRNAs for introduction of double-stranded DNA breaks in murine target sequences. Such breaks can be used for gene inactivation (via indels), small sequence insertion, or large sequence insertion. For sequence insertion projects, investigators must provide homology donor DNA. DNA encoding sgRNAs with or without homology donors will then be used for pronuclear injection of embryos from either C57BL/6 or CB6F1.

The fee for design, construction, and testing of sgRNAs is $1,500. This does not include the fee for pro-nuclear injection.

The reconstitution of live mice from cryopreserved sperm is accomplished via in vitro fertilization (IVF). This service includes the purchase price and super-ovulation of the egg-donor females, providing pseudopregnant foster dams for uterine transfers, per diems for pregnant embryo recipients, as well as the weaning and transfer of pups to the investigator. The core assesses a service fee of $1,500 for in vitro fertilization.

Rederivation is used to help eliminate pathogens from a mouse line. This may be required by the Division of Laboratory Animal Medicine if the health status of a desired strain is in question prior to its importation from another facility, or if a strain tests positive for a pathogen and elimination of the colony is the only other solution. Rederivation is accomplished by harvesting embryos from a group of females provided by the investigator. The isolated embryos are taken through a series of washes and then transferred into pseudopregnant foster dams. The foster dams are quarantined until their pups are weaned, and then testing is performed on the mothers to determine if the pathogen has been eliminated. The core assesses a service fee of $600 for rederivation. This includes the isolation and transfer of the embryos but does not include special fees for quarantine or the serological testing of the mothers.

When a mouse strain is not immediately needed, but may be needed later and cannot be readily obtained from an approved vendor, mouse sperm cryopreservation provides an efficient solution for backing up many genetically engineered mouse strains. Sperm cryopreservation can also protect mouse strains that are difficult to replace in the event of a pathogen outbreak, natural disaster, or cessation of your existing breeding stock. Sperm is isolated from two male mice between the ages of 3-6 months. Ideally, these would be males that have bred successfully in the past, but separated from the female for at least two weeks. Sperm is isolated and treated with a cryoprotective media that enables the germplasm to be stored indefinitely in liquid nitrogen. Because of the strain-to-strain variation in genetically engineered mice, we cannot always guarantee the success of this procedure. As a means of quality control, however, we routinely cryopreserve a test-straw to evaluate the viability, mobility and concentration of the frozen sperm. We are now including in the cost of sperm cryopreservation an additional step of quality control. We will use sperm from the test-straw to fertilize eggs and culture these embryos to the two-cell stage. If the percentage of eggs that go on to two-cell embryos is low, the investigator will be notified.

Sperm cryopreservation has three significant biological disadvantages compared to embryo cryopreservation:

1. Since a haploid genome is being frozen, more breeding of reconstituted mice may be necessary if more than one mutation and/or transgene is involved.
2. Some mutations must be maintained on a mixed or otherwise unusual background to maintain the desired phenotype, and oocyte donors with that background may not be available at the time of reconstitution.
3. As mitochondria are inherited exclusively from the maternal lineage, cryopreservation of sperm cannot be used to preserve strains of mice with mtDNA polymorphisms.

The core assesses a service fee of $650 for sperm cryopreservation. Long-term storage in liquid nitrogen is also included in this fee.

The Transgenic Mouse Core Facility generates transgenic mice via pronuclear microinjection of DNA constructs, including those for CRISPR-Cas9 projects, supplied by users. Microinjections are performed in embryos obtained from C57BL/6 or CB6F1 (a cross between BALB/c and C57BL/6) mice. After weaning potential founder mice, a small amount of tissue is taken from the tip of the tail and provided to users for genotyping. At that time each individual pup is permanently identified with an ear punch corresponding to the number it is given in the Transgenic Mouse Core Facility database. Users then identify mice harboring the transgene, usually by PCR amplification. To avoid costs, users must identify founders and initiate transfer of mice within three weeks. After that period, users will be charged a per diem rate of $2 per cage, per day. Please also note that production of transgenic mice will not be initiated unless an approved IACUC protocol is in place to allow transfer of the mice once they are identified.The core assesses a service fee of $4,000 for the generation of at least two transgene-positive mice.

The Transgenic Mouse Core Facility generates transgenic mice via pronuclear microinjection of DNA constructs, including those for CRISPR-Cas9 projects, supplied by users. Microinjections are performed in embryos obtained from C57BL/6 or CB6F1 (a cross between BALB/c and C57BL/6) mice. After weaning potential founder mice, a small amount of tissue is taken from the tip of the tail and provided to users for genotyping. At that time each individual pup is permanently identified with an ear punch corresponding to the number it is given in the Transgenic Mouse Core Facility database. Users then identify mice harboring the transgene, usually by PCR amplification. To avoid costs, users must identify founders and initiate transfer of mice within three weeks. After that period, users will be charged a per diem rate of $2 per cage, per day. Please also note that production of transgenic mice will not be initiated unless an approved IACUC protocol is in place to allow transfer of the mice once they are identified.The core assesses a service fee of $4,000 for the generation of at least two transgene-positive mice.

Core personnel will design and test sgRNAs for introduction of double-stranded DNA breaks in murine target sequences. Such breaks can be used for gene inactivation (via indels), small sequence insertion, or large sequence insertion. For sequence insertion projects, investigators must provide homology donor DNA. DNA encoding sgRNAs with or without homology donors will then be used for pronuclear injection of embryos from either C57BL/6 or CB6F1.

The fee for design, construction, and testing of sgRNAs is $1,500. This does not include the fee for pro-nuclear injection.

CRISPR-Cas9 can generate multiple indel mutations in a single mouse. This service will characterize the spectrum of indel alleles all mutants produced by pro-nuclear injection. PCR products from the modified locus will be amplified, cloned, and sequenced. The sequences will be aligned with the wild type allele and possible frame-shift and pre-mature stop codons identified.

When a mouse strain is not immediately needed, but may be needed later and cannot be readily obtained from an approved vendor, mouse sperm cryopreservation provides an efficient solution for backing up many genetically engineered mouse strains. Sperm cryopreservation can also protect mouse strains that are difficult to replace in the event of a pathogen outbreak, natural disaster, or cessation of your existing breeding stock. Sperm is isolated from two male mice between the ages of 3-6 months. Ideally, these would be males that have bred successfully in the past, but separated from the female for at least two weeks. Sperm is isolated and treated with a cryoprotective media that enables the germplasm to be stored indefinitely in liquid nitrogen. Because of the strain-to-strain variation in genetically engineered mice, we cannot always guarantee the success of this procedure. As a means of quality control, however, we routinely cryopreserve a test-straw to evaluate the viability, mobility and concentration of the frozen sperm. We are now including in the cost of sperm cryopreservation an additional step of quality control. We will use sperm from the test-straw to fertilize eggs and culture these embryos to the two-cell stage. If the percentage of eggs that go on to two-cell embryos is low, the investigator will be notified.

Sperm cryopreservation has three significant biological disadvantages compared to embryo cryopreservation:

1. Since a haploid genome is being frozen, more breeding of reconstituted mice may be necessary if more than one mutation and/or transgene is involved.
2. Some mutations must be maintained on a mixed or otherwise unusual background to maintain the desired phenotype, and oocyte donors with that background may not be available at the time of reconstitution.
3. As mitochondria are inherited exclusively from the maternal lineage, cryopreservation of sperm cannot be used to preserve strains of mice with mtDNA polymorphisms.

The core assesses a service fee of $650 for sperm cryopreservation. Long-term storage in liquid nitrogen is also included in this fee.

The reconstitution of live mice from cryopreserved sperm is accomplished via in vitro fertilization (IVF). This service includes the purchase price and super-ovulation of the egg-donor females, providing pseudopregnant foster dams for uterine transfers, per diems for pregnant embryo recipients, as well as the weaning and transfer of pups to the investigator. The core assesses a service fee of $1,500 for in vitro fertilization.

Rederivation is used to help eliminate pathogens from a mouse line. This may be required by the Division of Laboratory Animal Medicine if the health status of a desired strain is in question prior to its importation from another facility, or if a strain tests positive for a pathogen and elimination of the colony is the only other solution. Rederivation is accomplished by harvesting embryos from a group of females provided by the investigator. The isolated embryos are taken through a series of washes and then transferred into pseudopregnant foster dams. The foster dams are quarantined until their pups are weaned, and then testing is performed on the mothers to determine if the pathogen has been eliminated. The core assesses a service fee of $600 for rederivation. This includes the isolation and transfer of the embryos but does not include special fees for quarantine or the serological testing of the mothers.