Genetic Models Core

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.

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.

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.

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.