Provost
The overall goal of this facility is to provide the means by which researchers can learn and apply cutting edge technology to the molecular analysis of mammalian gene function. The specific aim of this facility is to create "knockout mice" through the utilization of DNA-, stem cell-, and embryo-manipulation procedures. Molecular cloning techniques are employed to clone and manipulate DNA sequences. Homologous DNA recombination in cultured embryonic stem (ES) cells is employed to generate "targeted" ES cells (i.e. ES cells carrying the replacement of specific chromosomal DNA sequences with cloned DNA sequences). Embryo manipulation techniques involving the targeted ES cells are employed to generate chimeric mice, which are then used to generate the knockout mice.
| Demetri Spyropoulos, Ph.D. | Director |
| Yong Z. Gong, M.D. | Embryo manipulation |
| Tina Cooper | Embryonic stem cell culture |
PCR ID of Cre-Recombinant ES cells
Investigators can use the Gene Targeting & Knockout Mouse Facility to generate mice carrying any sequence-specific genetic modification (ranging in size from 1bp to over 100 Kbp). The mutation can be the deletion of an entire locus of genes, a specific reciprocal chromosomal translocation, a complete gene knockout, a domain-specific gene knockout (or alteration), or a promoter/enhancer knockout (or alteration). The mutation can be made conditional using, for example, a Cre-recombinase based strategy. The mutation can be a "knock-in," involving the replacement of one genetic element for another. Targeted genes can also be made to express other genes, such as genetic markers.
The Facility has demonstrated expertise in the generation of knockout mice, cre-conditional knockout mice and sequence-specific mutant mice. The Facility has also demonstrated the ability to generate non-chimeric, entirely ES cell-derived mice and cre-recombinant knockout mice via infection of targeted ES cells with adenovirus that express cre recombinase. With these capabilities, the Facility can provide a majority of the "cutting-edge" technologies available in the field of knockout mouse technology, the most notable of which are tissue-specific inducible genetic alterations.
Embryo cryopreservation has also been established as a service for the storage of mouse strains. This service was made possible through funding for the purchase of a controlled-rate freezer, supported by the URC. The embryo cryopreservation service will help accommodate limitations in animal space, mouse colony contamination, increases in the number of researchers using small rodents, and increases in the number of small rodents used by individual researchers. The Facility has demonstrated expertise in the cryopreservation of mouse embryos and in the utilization of cryopreserved embryos for the generation of chimeric mice.
The facility director will provide the guidance for the synthesis of gene targeting vectors. The facility will provide for the generation of 96 selected ES cell lines, the generation of chimeric males, and technical guidance for the characterization of offspring that are heterozygous and homozygous for the targeted mutation. We do not guarantee that targeted cell lines and germline chimeras will be generated, however, we will provide documentation for the targeting efficiencies (using other targeting vector constructs) and the germline transmission efficiency for the starting ES cell line. ES cells will be tested periodically for mycoplasma, karyotype, targeting efficiency, and germline transmission. New ES cell lines, derived in this facility will be tested according to these criteria and thus allow optimal germline transmission, even when extensive manipulation in culture is required. Consultation on targeting vector constructs and the analysis of knockout offspring will maximize time and cost efficiency, as well as provide the user with the most recent advances in targeted mutagenesis and chimera analysis.
This facility occupies 1500 sq ft of the Hollings Cancer Center and contains two 6 ft Baker SterilGARD Class II Hoods, two Forma CO2 incubators, one Nikon dissecting scope equipped with a Hamamatsu videocamera on a Vibraplane isolation table, one Zeiss Axiovert 10 injection scope with cooling stage and Narishige Micromanipulators on a TMC isolation table, an RM6 Lauda cooling water bath, one Leitz Labovert ES cell scope equipped with a Leica photomicrographic system, a Biorad Gene Pulser, a Taylor-Wharton cryostorage unit, a Planar Controlled Rate freezer for embryo cryopreservation, a Holton LaminAir dual workstation hood, water baths, and a refrigerator/freezer. The animal facility is a Specific Pathogen-Free facility that occupies 20,000 sq ft, contains 24 rooms, animal husbandry personnel, and houses cage washers, autoclaves, isolations cubicles, and micro isolation cages. The facility also boasts a new blastocyst injection station. The state-of-the-art components of this station are arguably among the best in the nation. These include a high-powered inverted microscope with optical capabilities for clearly observing mouse blastocyst stage embryos and embryonic stem cells simultaneously through glass and plastic dishes (DIC and Hoffman contrast), full-capability micromanipulator equipment for gently handling and injecting blastocyst stage embryos with embryonic stem cells while maintaining viability of both (micromanipulators, injecting and holding pipettes, piezoelectric manipulator; cooling waterbath and cooling stage); and the ability for other personnel (director, trainees, etc.) to observe the procedure (video camera, monitor, and recorder). Of note, the piezoelectric manipulator will allow for minimal damage to blastocyst embryos during injection and can be applied to techniques of Nuclear Transfer and ICSI. Such a blastocyst injection station will significantly increase the efficiency and throughput of knockout mouse production, especially because 1) most embryonic stem cell lines are more amenable to blastocyst injection than to morula aggregation, and 2) blastocyst injection requires fewer manipulation and shorter incubation times outside of the female reproductive tract than does morula aggregation.
- Mouse tail DNA Preparation
- $2/tail
We receive the tail sample in a 1.5ml eppendorf tube, add 400µl of Lysis Buffer. The sample is digested overnight at 55°C with 200µg Proteinase K. The next day 200µl of saturated NaCl is added and the sample is shaken vigorously and placed on ice for 10 min. The sample is then centrifuged for 10 min. and 500µl of the supernatant is transferred to 1ml of 95% ETOH. The DNA is spooled and placed in 50µl TE. Concentrations are determined by spectrophotometer. - ES Cell DNA Preparation
- $5/Targeted ES Cell Colony
The targeted ES cell colony is plated on a gel coated 96-well TC plate and allowed to grow to over-confluency (3-4 days). The well is rinsed with 1X PBS then 400µl of Lysis Buffer is added and the cells collected into a 1.5ml eppendorf tube. The sample is digested for 4 hours at 37°C with 100µg Proteinase K. 200µl of saturated NaCl is added and the sample is shaken vigorously and placed on ice for 10 min. The sample is then centrifuged for 10 min. and 500µl of the supernatant is transferred to 1ml of 95% ETOH. The DNA is spooled and placed in 25µl TE. Concentrations are determined by spectrophotometer. - Mouse Genotyping
- $125/25 PCR
Reactions The tail sample is received in a 1.5ml eppendorf tube, 200µl of Viagen PCR Lysis Buffer is added. The sample is digested overnight at 55°C with 200ug Proteinase K. The next day, the sample is heat-inactivated at 90°C for 10 min. then placed on ice. The PCR reactions are set-up (Promega 2X PCR kit or customized PCR reaction) with the PI-supplied primers. The samples are run on a 2% agarose gel with 100bp ladder. Pictures are taken of the gel and supplied to the PI. - ES Cell Electroporation
- $5,500/96 Colonies
Targeted Vector DNA is supplied by the PI with 10-14 kb homology. The DNA is electroporated into ES cells and plated onto 10cm TC dishes with feeder layer. The ES cells fed daily with drug-selection media and are allowed to grow until colonies are large enough to pick into 96-well TC feeder plates (8-10 days). Once colonies are picked, they are continued to be fed with drug-selection media daily for 2 days. The colonies are trypsinized on the 3rd day onto new 96-well TC feeder plates. They are fed daily with drug-selection media for 2 more days. The colonies are again trypsinized on the 3rd day and half of the cells are transferred to gel-coated 96-well plate designated for DNA collection and the other half of the cells are frozen at -80°C. The DNA plate is fed daily with drug-selection media and allowed to grow to over-confluency. The wells are rinsed with 1X PBS and 200µl of Lysis Buffer is added. The lysed ES cells are collected into 1.5ml eppendorf tubes and 100µg of Proteinase K are added. The samples are digested at 37°C for 4 hours. The samples are then given to the PI for further analysis. Once targets are determined by Southern Blotting, the targeted ES cell colonies are thawed and transferred to a new 96-well TC feeder plate. The colonies are fed daily with drug-selection media, trypsinized every third day to prevent differentiation, and two vials of each colony are frozen away. - Blastocyst Injection
- $2,200 to produce 2 Chimeras (>20%) in-house
$2,500 to produce 2 Chimeras (>20%) outside
Day 1 - four C57 BL/6 female mice are injected with PMSG to superovulate to provide 50-100 blastocysts. Day 3- the same females are injected with HCG and mated with 4 breeder males. Targeted ES cell colonies are thawed onto 1-10cm feeder dish. (Cells are fed daily). Day 4 - 2-4 CD 1 females are mated with 2-4 vasectomized males to create foster moms. Day 6 . the C57 BL/6 females. uteri are flushed and blastocysts are collected. Targeted ES cell colonies are trypsinized into single cell suspension. The collected blastocysts are micro-injected with the single cell targeted ES cells. The micro-injected blastocysts are implanted into uteri of the foster moms. Day 23-24 . foster moms give birth to implanted pups. If foster moms have not given birth by Day 24, a c-section is performed. Day 31 . coat color of pups can be seen and chimera % determined.
Blastocyst injection
Morula aggregation - Embryo Cryopreservation
- $100 set-up fee + $50 (includes first 20 embryos) + $7.50/additional embryo.
$250 yearly storage fee.
The embryos are collected by flushing from females provided by PI. The embryos are placed in a 35mm TC dish and the first freezing media is added for 5 min.at room temp. The embryos are transferred individually to a second freezing media for 10 min. at room temp. Then the embryos are placed in a third freezing media for another 10 min. The embryos are placed in a cryovial and put into the Planer Kryo Biological Freezer to initiate cooling. The embryos are a frozen over a period of 3 hours to a final temp. of -35°C. The cryovials are transferred to liquid nitrogen for long-term storage.
Cryopreservation Facility - Blastocyst Transfer
-
$600/litter of 4 pups + cryopreservation charges if
required.
$300/litter of 1-3 pups + cryopreservation charges if required.
Day 1 . 2-4 CD1 females are mated with 2-4 vasectomized males to create foster moms. Day 3 embryos are collected by flushing from females provided by PI. The embryos are implanted into the uteri of the foster moms. Day 23-24 . foster moms give birth to implanted pups. If foster moms have not given birth by Day 24, a c-section is performed. Day 45-46. the pups will be weaned and returned to PI. - Chimera Maintenance and Breeding
- $75/Chimera/month (includes mating females)
2 mating females (CD1 or BL/6) are placed into cage with chimera males and housing costs of all mice are included for the month. These prices will also apply to Germline Chimera Colony production, however the PI is advised to transfer animals prior to this point. (PI.s should provide the facility with the ARC # at the time of germline indentification.) - Mouse Weaning and Tailing
- $20/litter/month
The pups are placed into new cages when mature (3 weeks) and identifications are made for each. The pups' tails are cut and collected into individual 1.5ml eppendorf tubes. - Timed Matings
- $30/hour
The price reflects tech time to set-up matings, check plugs, and monitor embryo development. - Various Mouse Surgical Procedures
- $50/mouse/procedure
Procedures include dissection of pups from timed matings, MEF production, and other procedures as requested. - Knockout Offspring Maintenance
- $12/mouse/month (after weaning)
The price reflects all housing costs for Knockout Offspring. - Mouse Purchase
- CD1 $35/1 mouse + $4.00 for each additional mouse; BL6 $75/1 mouse + $15 for each additional mouse. Includes: Cost of mouse without paying shipping costs. We suggest PI.s purchase animals directly from vendor.
- Breeding Mice
- Charge for mouse purchased + 5 weeks (until maturity) maintenance
fee ($15) Includes:
- CD 1 or BL/6 mice for breeding
- All housing fees incurred until breeding mouse is mature.
Electroporation Request Form
In addition to this form, we request 100 micrograms of linearized vector DNA,
containing a total of approximately 10 Kb of homologous isogenic (mouse strain
129/Sv) sequences flanking the positive selectable marker. The DNA sample
should be sterile, of high purity and at a concentration of approximately
1mg/mL.