Transgenic Mouse

 

transgenic mouse

Shared Resource Director: Stephen F. Konieczny
Facility Director: Judy Hallett
E-mail: jhallett@bilbo.bio.purdue.edu
Department: Biological Sciences
Phone: (765) 496-3352
Building/Room: HANS, Room B028
 

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General Information and Services

The Transgenic Mouse Core Facility (TMCF) offers a variety of services to the Purdue University Center for Cancer Research community, including (1) generation of transgenic mice, (2) gene targeting in embryonic stem cells, (3) blastocyst injections for the generation of knock-out mice, (4) morula aggregation for chimeric mouse production, (5) strain rederivation, (6) in vitro fertilization, (7) embryo cryopreservation, (8) sperm freezing, (9) intracytoplasmic sperm injection and (10) mouse embryo fibroblast (MEF) cell production.

Each service is described below in detail. For additional information please visit the TMCF website 

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Overview

To initiate a request for any of our services, each principal investigator submits an animal protocol to the Purdue University Animal Care and Use Committee (PACUC) for approval. Once the protocol is approved and the investigator is assigned a PACUC number, the principal investigator is ready to make a formal request to the TMCF. This request is initiated by filling out an appropriate order form (available on the TMCF website ) for each particular service. These forms are presented at the initial strategy meeting attended by the principal investigator, the lab contact person and the TMCF Manager and Scientific Director. The lab contact person is particularly important since the TMCF Manager updates each project on a weekly basis. The lab contact person is responsible for providing the specific DNA for ES cell electroporation or pronuclear injections, receiving the ES cell lines or tail DNA for analysis, transferring the positive founders or chimeras to the principal investigator’s animal space, and reporting all results from analysis or breeding of the mice back to the facility. A summary of the injection and embryo transfer sessions performed by the TMCF is provided with each set of tail DNAs or chimeric pups delivered to the contact person. Protocols and advice on analysis and breeding of generated mice is readily available from the TMCF. Training in handling, sexing, tailing and marking mice also is available upon request.

For transgenic mouse production, the TMCF typically performs three injection sessions. For chimeric mouse production, two sets of three injections (or morula aggregations) are performed. Re-injections are done at the TMCF Manager’s discretion. If there are no founders or chimeras from the initial injections, the TMCF may conclude that the problem lies with the construct, the DNA preparation, or the ES cell clones, and re-injection will only be performed after submission of a new injection request. If there is a technical problem associated with the TMCF itself, additional injection series will be performed at no charge to the investigator.

Production of Transgenic Mice

DNA preparation is critical to the success of any transgenic experiment. Therefore, we attempt to minimize problems by having the TMCF perform the actual purification steps. Briefly, investigators provide a complete restriction digest with a portion of the sample separated by gel electrophoresis and documentation showing the correct fragment to be isolated. The TMCF then runs a similar gel and purifies the appropriate DNA fragment by standard procedures.

The usual strain of mouse used for transgenic injections is B6CBA, an F1 hybrid of C57Bl/6 and CBA/J, ordered from Harlan. This is a hardy agouti hybrid, easily superovulated, and commonly used for microinjection. The resultant pups will be an F2, B6CBA X B6CBA cross and can be agouti or black. The TMCF also uses novel strains for microinjection sessions upon request. The cost of obtaining these animals is charged to the investigator. Once injections have been completed and embryos transferred to foster mothers, the facility provides the necessary care to the recipient females and subsequent pups until weaning. Tail samples from potential transgenic litters are then taken at 21 days of age and provided to the lab contact person who is responsible for performing standard genotyping. At this stage transgenic pups are turned over to the lab contact.

Electroporation of ES Cells

The TMCF offers the option of performing the electroporation of ES cells with a specific targeting vector and the isolation of appropriate drug resistant cell lines. Individual clones (minimum of 300) are then returned to the principal investigator who is responsible for establishing which cell lines contain true targeted disruptions of the gene of interest. Alternatively, principal investigators may choose to generate their own targeted ES cell lines and then provide the cells to the TMCF for blastocyst injections (or morula aggregation). Prior to blastocyst injection, the TMCF requires evidence that the submitted ES cells are in excellent condition. This evidence consists of karyotype analysis of the targeted clones(s), mycoplasma testing, and cellular morphology.

Blastocyst Injections or Morula Aggregation

The TMCF injects targeted ES cells into blastocysts derived from superovulated C57Bl/6 females and transfers the blastocysts into pseudopregnant CD1 females. Alternatively, morulae are collected from similarly superovulated C57Bl/6 females, aggregated with targeted ES cells and transferred to pseudopregnant CD1 females. Following blastocyst injection or morula aggregation, recipient females are cared for by the TMCF until pups are weaned and an obvious coat color is apparent. Chimeric pups are then transferred to the lab contact person who is responsible for additional matings to establish germ-line transmission of the targeted allele.

Strain Rederivation

In order to allow investigators the ability to house pathogen-free animals obtained from other sources (typically from collaborators at other institutions) in our animal facilities, embryo rederivation can be performed. Timed pregnant mice (2 days for embryo transfer, 18 days for Caesarian section) are shipped to Purdue on a pre-arranged day and sacrificed immediately upon arrival in a separate quarantine laboratory. The entire oviduct/uterus is removed, washed with disinfectant solution and brought to the TMCF in a sterile petri dish where the embryos are harvested and transferred into pseudopregnant recipient mice or the pups fostered out to new mothers. Once pregnancy or pup acceptance has been confirmed, the foster mothers are transferred to the lab contact person as described above, who is responsible for genotyping the individual mice.

In Vitro Fertilization

Under some circumstances it is necessary to perform in vitro fertilization to ensure that a particular trait (i.e. transgene or targeted allele) is passed on to progeny. A male mouse, usually with poor reproductive history and of great genetic value, is sacrificed and sperm collected from epididymes and vas deferentia. Oocytes collected from superovulated wild type females are incubated with the sperm and fertilized embryos are transferred to oviducts of pseudopregnant CD1 females.

Embryo Cryopreservation

In order to maintain back-up animals as well as to keep the cost of housing mice to a minimum for individual investigators, the TMCF offers an embryo cryopreservation service. Morulae are flushed from the oviducts of superovulated wild type females who have mated with males of genetic value. These embryos are frozen to -40°C at a rate of -1°C/min and stored in liquid N2. The frozen embryos remain viable indefinitely and may be thawed and rederived as described above.

Additional Services Offered by the TMCF

The TMCF has recently introduced several new services that are offered to the Purdue Cancer Research Center community. These include (1) sperm freezing for storage of valuable mouse lines, (2) intracytoplasmic sperm injection for the production of mice from males with poor mating records or with low motility sperm and (3) mouse embryo fibroblast production from any mouse line for in vitro experiments that are carried out by the investigator. Please contact the TMCF for more information concerning these new services.

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