Transgenic Mice
Shared Resource Director: Stephen
F. Konieczny
Facility Director: Judy Hallett
E-mail: jhallett@bilbo.bio.purdue.edu
Department: Biological Sciences
Phone: 496-3352
Building/Room: HANS, Room B028
General Information and Services
The Transgenic Mouse Core Facility (TMCF) offers
a variety of services to the Purdue Cancer Center 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 web site at http://www.bio.purdue.edu/research/groups/tmcf/index.htm
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 Web
Site at http://www.bio.purdue.edu/research/groups/tmcf/index.htm)
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 then 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) then
are 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 before injections are
initiated. 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 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.
Transgenic Mouse Rates
(Rates for Cancer Center members will be discounted)
Production of a Mouse: $2,850
ES cell target knock out: $9,970
Blastocyst injection only: $4,800
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