- CRISPR/Cas9 system and ES Cell Gene Targeting System to Generate Mouse Models with a Site-specific Genome modification
- Mouse Transgenesis by Random Integrations
- Specific Pathogen Free Mouse Line Rederivation
- Mouse Embryo/Sperm Cryopreservation
- Mouse Line Recovery from Cryopreserved Embryos/Sperm
- In Vitro Fertilization (IVF) with Fresh or Frozen Mouse Sperm
- Blastocyst Injection with ES Cells (Chimeric Mouse Production)
- Mouse Intracytoplasmic Sperm Injection (ICSI)
- De Novo Generation of Mouse ES Cell Line
- Genotype Analysis
- Colony Scale-Up
CRISPR/Cas9 System: The Brown Mouse Transgenic and Gene Targeting Facility is providing full services of de novo generating genetically modified mouse models using cutting edge technologies of CRISPR/Cas9 system. These mouse models can be made of a gene knockout, in-frame deletion, conditional knockout, gene point mutation, gene tagging, as well as gene replacement. The full services include molecular design, guide RNA validation, donor plasmid construction, single stranded DNA template production, embryo microinjection or electroporation, embryo transfer, founder mouse identification, and confirmation of founder mouse germline transmission. Although wild type C57bl/6J or C57bl/6N inbreed mice will be routinely used for the model production, other strain of wild type mice, either inbreed or hybrid such as CD1, CF1, or FVB/N can be applied too. In addition, further modification on a particular transgenic mouse line with clear genetic background provided by the investigator can be made in the Facility. Normally, the whole process with CRISPR/Cas9 system from guide RNA validation to F1 delivery takes 6 to 8 months. A minimum of two F1 heterozygous mice (either males or females) will be delivered to the investigator.
ES Cell Gene Targeting System: Despite vast majority of the projects can be done with CRISPR/Cas9 system, projects requiring site-specific knock-in of a relatively large DNA sequence (eg, >3 kb) into the mouse genome will be done through conventional ES cell gene targeting system. This process compared to the CRISPR/Cas9 procedure will take extra time (normally 3 to 6 months) at the steps of gene-targeting vector construction and establishment of the gene-targeted ES cell lines that will be used for ES cell injection into wild type morula or blastocysts to make chimeric mice (as the F0 founders). These steps can be done in the investigator’s lab or in the facility. If ES cells are targeted by the facility, the investigator will provide the purified construct (~2 μg/μl, >100 μg in 10mM Tris-HCl, 1.0mM EDTA, pH7.5) for electroporation. The investigator will be charged for electroporation, selection, and archiving of ES cell clones, in addition to the injection of ES cells into embryos. We will inject and implant a minimum of 60 ES cell-injected blastocysts. If the gene-targeted ES cell clones were generated in the investigator’s lab or facility, three males with a chimeric coat (B6 ES cells, > 50%; 129 ES cells, >75%) are guaranteed.
For an additional fee, the facility will breed the generated chimeras to test germline transmission. A maximum of five Chimeric males will be bred to wild type females. Plugs will be checked daily and new females will be rotated into each male's cage when a plugged female is removed. This will continue until the germline transmission is confirmed, or all males have produced 50 negative pups, or the males are 8 months old.
Transgenesis by Random Integrations: DNA fragments or circular bacterial artificial chromosome (BAC) can be injected into mouse pronucleus to make transgenic mice with random integrations in the mouse genome. The investigator will provide the construct sequence and endotoxin-free plasmid (> 200 ng/μl, > 40 μl in 10mM Tris-HCl, pH8.5) purified by an endotoxin-free preparation kit (QIAGEN or MACHEREY-NAGEL) or BAC (please contact transgenic facility for BAC preparation). The transgenic facility will do restriction enzyme digestion for a gel purification of the DNA fragments that will be injected for random integrations in mouse genome. This service can be applied to the mouse strain of CD1, C57bl/6, FVB/N, or the strain provided by an investigator. Transgenic facility guarantees to deliver a minimum of three founders with DNA integrations, but no guarantee for the transgene expression from any founder due to the complexity of random integrations.
Rederivation: Transgenic facility provides in-house rederivation services from live animals. All mouse imports to Brown University need to be approved by the animal care facility (ACF) authority. After approval, 5 to 10 male mice at ages of 2-10 months old will be transferred from the investigator’s animal room to the quarantine room by ACF staffs. The transgenic facility will order appropriate wild type females that will be superovulated and mated to the provided males in the quarantine room. The harvested embryos will be thoroughly washed and implanted into specific pathogen free (SPF) pseudo-pregnant females that will be housed at the transgenic facility quarantine room/rack. After pups are born, the tissues for genotyping will be collected at 7 to 10 days old. Genotyping will be responsible by the investigator's lab, or by transgenic facility for an extra charge. When the derived pups have reached weaning age, ACF staffs will help to collect feces and prepare body swabs from the foster moms for health testing. All quarantine costs, including health testing, are the responsibility of the investigator. Two rounds of superovulation will be performed. Generally, 10 to 20 progeny are produced depending on the fertility of the males provided.
Cryopreservation: The mice provided by investigators will be clearly marked on their cage cards with a description of “for Cryo”, project/gene name, animal ID (if available). (1) Heterozygous embryos. For each mouse line, the investigator provides 6-10 male mice (at least 8 weeks old) that are single-caged in the investigator’s animal room. Transgenic facility will order appropriate females for superovulation and mating. The oviducts at day 1.5 post coitum will be harvested by the facility staff in a biosafety cabinet and transported to transgenic facility in cold M2 medium. Embryos at the 2-4-cell embryo stage will be flushed out from oviducts and cryopreserved. Normally, this procedure will be performed twice to cryopreserve a minimum of 120 embryos in 4-5 straws (25 to 30 embryos/straw). (2) Homozygous embryos. Besides 6-10 male mice over 8 weeks old, the investigator will provide 10 to 20 females (from the same line as the males) that are 3 to 6 weeks old (not mated with males after wean). The procedure will be the same as the heterozygous embryo cryopreservation. A minimum of 120 embryos will be cryopreserved with enough females provided by the investigator. (3) Sperm. For each mouse line, the investigator will provide 2 to 3 healthy males at 3-10 months old. At least one cauda epididymis will be harvested in a biosafety cabinet from each male for sperm isolation, and a mixture of sperm from all males will be cryopreserved in a form of 10μl of sperm per straw for a total of 10 straws per mouse line. The embryos or sperm will be stored in a liquid nitrogen tank. Annual storage fee will be charged starting from the date when the cryopreservation was done.
Mouse Line Recovery from Cryopreservation: (1) from frozen Embryos. Embryos stored in Brown transgenic facility or imported from other institutions can be thawed and implanted to pseudo-pregnant females in order to recover the frozen mouse lines. All embryo imports must be approved before shipping to Brown transgenic facility by the ACF authority. All cost from shipping and handling, quarantine, and health testing are the responsibility of the investigator. Samples will be collected from pups at 7 to 10 days after birth by transgenic facility staffs. Genotyping will be done in the investigator’s lab without a cost or in the transgenic facility for an extra fee. Internal transfer of mice to the investigator will be done after weaning of mice. Normally, two implantations with 20 to 30 embryos per line will be done and a minimum of 4 mice will be delivered to the investigator. (2) from frozen sperm. Refer to IVF with frozen sperm procedure.
In Vitro Fertilization (IVF): Success depends greatly on the sample of sperm provided and the background strain requested. (1) with fresh sperm. The investigator will provide the male mice (one or multiple). Cauda epididymis will be harvested in a biosafety cabinet and put in a 200μl PCR tube containing cold storage medium, then transported in a cold kit to the transgenic facility. The sperm will be isolated from epididymis on the same day or after overnight storage in a refrigerator for IVF. Ten wild type females at 3-4 weeks old will be ordered and superovulated to provide oocytes for IVF. Pseudo-pregnant CD1 or SW mice will be prepared for embryo implantation. IVF and embryo transfer will be done in the transgenic facility. Samples will be collected from pups at 7 to 10 days after birth by transgenic facility staffs. Genotyping will be done in the investigator’s lab without a cost or in the transgenic facility for an extra fee. Internal transfer of mice to the investigator will be done after weaning of mice. Generally, 10 to 30 progeny are produced depending on the fertility of the males provided. (2) with frozen sperm. Sperm stored in Brown transgenic facility or imported from other institutions can be thawed for IVF. Other operations are the same as IVF with fresh sperm. Generally, 10 to 20 progeny are produced depending on the fertility of the thawed sperm.
Chimeric Mouse Production: The investigator provides gene targeted ES cells for injection. The transgenic facility provides services to make chimeric founders and germline transmission test. Three males with a chimeric coat (B6 ES cells, > 50%; 129 ES cells, >75%) are guaranteed. Please contact us for a protocol “ES cell culture for injection”.
Colony Scale-Up: Investigators who need to quickly expand a colony can use our scale-up service to do this quickly and reliably. The investigator provides 6-10 homozygous male mice to be mated with superovulated wild type females. This produces heterozygous embryos to be implanted. When the heterozygous pups are born, the females will be superovulated and bred to the same homozygous males. This 2-step process will produce large numbers of homozygous and heterozygous mice. Other breeding strategies may be used to suit the investigator's needs. This service is available only for mice housed in the Ship Street facility.
For further information or if you require services that are not listed here, please contact Jinping Luo ([email protected]), the facility director.