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To generate knock-out or knock-in mice, both the gene targeting (see below) in mouse embryonic stem (ES) cells and the mouse blastocyst microinjection (see below) procedures are required. The total cost of the two procedures is $10,400.
Overview of Procedures: Gene-targeted mouse ES cells are produced by electroporating a gene targeting construct into wild-type ES cells. The targeting construct typically contains selectable markers surrounded by two regions of homology to the targeted locus. In a fraction of those cells that take up the targeting construct, homologous recombination between the genome of the ES cell and the two regions of homology will result in the replacement of the targeted locus with the targeting construct.
PCR and/or Southern blotting can then be employed to identify those ES cells that have correctly incorporated the targeting construct into the genome. Generally, the gene-targeted ES cells will be hemizygous for the targeting construct.
 Knock-out or knock-in mice are created by microinjecting these gene-targeted mouse ES cells into mouse blastocysts. The injected blastocysts are then transferred to pseudopregnant females and allowed to develop to term. After pups are born, some of them may be chimeric (i.e. they will have one or more tissues to which the targeted ES cells have contributed).
After reaching sexual maturity, the chimeric mice are bred to appropriate wild-type mice and the resulting pups are screened for the presence of heterozygous targeted allele if the targeted ES cells have participated in germline formation of the chimeras. Further heterozygote breeding is required to generate mice that are homozygous for the targeted locus (knock-out mice).
Gene Targeting in Mouse Embryonic Stem Cells
The Transgenic Mouse Core Facility staff electroporates linearized gene-targeting constructs into wild-type 129SvEv mouse embryonic stem (ES) cells, and then cultures electroporated ES cells in selection media. After selection, we will pick up three 96-well plates of colonies, freeze the stock plates and provide replica plates to the investigator for DNA isolation. Targeted ES cell clones will be determined by the investigator, and thereafter will be expanded by the Core Facility for blastocyst microinjection. The fee for this procedure is $6,300 per electroporation.
Criteria for Gene Targeting Constructs
· All investigators must use a vector with a known map showing sizes and cloning sites of arms of homology, site of linearization, and selection cassettes. The Core Facility staff can provide you with an approved vector and map free of charge.
· The investigator must use isogenic genomic DNA for the creation of homologous arms. The Core Facility staff can provide you with appropriate genomic DNA.
· Short arms must be a minimum of 1 Kb in length, and the long arms a minimum of 3 Kb in length.
· Sequencing both arms, inserts, and all junctions is strongly recommended.
· The investigator must provide evidence that 3’ and 5’ arms are each in the correct orientation.
· The investigator must run a pilot experiment to test the screening strategies. Screening by Southern blotting is strongly recommended.
When the construct is completed, a meeting with lab personnel is required before delivery of the construct. Please bring the following to this meeting:
· Completed Gene Targeting Request Form.
· Copy of the vector map.
· Copy of Southern blot results.
· A figure showing the wild-type allele of your targeted gene, gene targeting construct, and targeted allele in linear form. Restriction sites used for Southern analysis, the location of Southern probe(s), and PCR primers used for screening must be depicted on this figure.
· Gel photo comparing the linearized construct to the circular plasmid.
Scheduling Requirements for Gene Targeting
· Services will not be scheduled until all project criteria are met and the Gene Targeting Request Form is approved.
· Upon acceptance of the above, your construct will be added to the project queue. At that time, you can provide us with 100 µg of the linearized construct in 1 mL of 70% ethanol.
· At least seven days advance notice will be required for the cancellation of a project, otherwise a cancellation fee will be charged to cover any costs incurred due to the cancellation (e.g., for ES cell setup in anticipation of the project).
· Services will be provided on a first-come, first-served basis, and a tentative date for initiation of services will be provided upon the approval of the Gene Targeting Request Form.
Embryonic Stem Cell Blastocyst Injection
The Transgenic Mouse Core Facility staff microinjects gene-targeted ES cells into about 200 C57BL/6 blastocysts for each construct, implants the injected blastocysts into10-15 pseudopregnant females, and takes care of the pregnant females and their litters up to 2 weeks after the pups are born. At this time the pseudopregnant mothers and their chimeric pups are transferred to the investigator for breeding. The Core Facility will generate at least three mid- to high-percentage chimeric pups if the ES cells meet the criteria below. The fee for this procedure is $4,100 per construct.
Criteria for Embryonic Stem Cells Provided by Investigators
· The investigator must provide the information about the origin and passage number of the ES cells.
· 3 positive ES cell clones for each construct are recommended.
· The ES cells must be free of bacteria on the injection day, and have been tested negative for mycoplasma within the last 6 months.
· A procedure to remove feeder cells must be performed before sending ES cells for blastocyst injection. A detailed protocol is available from the Core Facility staff.
o Core Facility personnel can prepare the targeted ES cells for blastocyst injection at a charge of $400 per preparation.
· The ES cells must be acceptable as judged by the microinjectionist.
Scheduling Requirements for ES Cell Injections
· Services cannot be scheduled until all project criteria are met and the Blastocyst Microinjection Request Form is approved.
· Prior to the initiation of any Blastocyst Injection service, all investigators are required to have a proven screening strategy and to obtain an approved active animal protocol justifying the particular experiment. The Transgenic Mouse Core Facility will not initiate any service without a valid animal use protocol number. Please visit the Animal Care and Use Committee website for more information about obtaining an animal use protocol number.
· ES cells provided by the investigator and judged to be in unacceptable condition by the microinjectionist will not be injected, and the investigator will be charged for the cost of the mice and any supplies used in the collection of blastocysts for microinjection.
· At least 14 days advance notice will be required for the cancellation of a project, otherwise a cancellation fee will be charged to cover any costs incurred due to the cancellation (e.g., for donor mice already purchased in anticipation of the project).
· Services will be provided on a first-come, first-served basis, and a tentative date for initiation of services will be provided upon the approval of the Blastocyst Microinjection Request Form.
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