Gene targeting is a technique utilizing homologous recombination between an
engineered exogenous DNA fragment and the genome of the mouse embryonic
stem (ES) cells. Recombination between identical regions contained within
the introduced DNA fragment and the native chromosome will lead to the
replacement of a portion of the chromosome with the engineered DNA. These
modified ES cells can then be injected into mouse blastocysts where they
can incorporate and contribute to the fetal development along with the
blastomeres from the ICM (inner cell mass). These techniques can be used
to ablate (knockout) gene function throughout the mouse, in selected tissues,
or at specific time points of mouse development. They can also
be used to introduce mutations into the genome at a desired location.
all gene targeting experiments have the following steps:
Depending on your needs, the Transgenic Animal Facility is able to perform any or all of the steps listed above: planning and construction of gene targeting vectors, targeting ES cells or blastocyst injection.
proceeding with any steps (Vector Design, Gene Targeting or Microinjection)
the investigator will need to meet with the Transgenic Animal Facility.
This consultation will enable us to determine the specific needs of the
investigator, review the targeting procedure and discuss fees for the
various services that the investigator wishes the GEAM to provide. Please
contact Kathy Krentz (firstname.lastname@example.org),
(608) 890-3785 to schedule an initial meeting.
Gene Targeting Vectors
for Vector Design
recommend incorporating the following features in your targeting vector.
Custom Design or Production of Targeting Vector
staff expert in molecular biology techniques can construct targeting vectors
for your project for a time and material recovery fee. We will work
with you to design a vector suitable for your experiment using the guidelines
described above in Guidelines for
Vector Design. We have designed and produced knock-out,
knock-in, and conditional knock-out vectors for a number of UW-Madison
to scheduling your targeting: You will
targeting vector will be introduced by electroporation into R1 embryonic
stem cells (Nagy et
al., 1993) ES cells that integrate the targeting vector either by homologous
or random integration will be selected by growth on G418. Negative
selection with gancyclovir will select against clones that contained the
HSV-TK cassette. This will enrich for clones that integrated the
Neo cassette by homologous recombination, and select against random integration
events. Up to 480 Neor, GANCr colonies will be picked, duplicated and
frozen in 96-well dishes.
will be prepared from the duplicated 96-well plates and given to the investigator
for genotyping. We highly recommend using Southern blotting with
probes that lie 5' and 3' to the regions of homology included in the targeting
vector. The investigator will also be responsible for verification,
by DNA sequence analysis, of any loxP sites, FRT sites or specific mutations
introduced by the targeting vector.
targeted clones will be amplified, frozen in cryovials, and stored in
liquid nitrogen. A sample of genomic DNA will be made from
each clone and given to the investigator for re-genotyping and sequencing
the clones have been reconfirmed by Southern blot we recommend karyotyping
be done to identify ES clones with a normal chromosome number. We
currently subcontract this service. Karyotypically normal ES clones
are ready for microinjection.
Microinjection of ES Cells
you are ready to have your ES cell clones injected: You will need
ES cells are grown for three to four days on a LIF (leukemia inhibitory
factor) producing feeder layer. ES cells are disaggregated into
single-cell suspension, separated from the feeder cells, and injected
into the blastocoel cavities of expanded blastocysts. At this point,
since most investigators choose to use ES cells derived from Strain 129/Sv
mice to generate their knockouts, the ES cells are injected into C57BL/6
blastocysts which have been demonstrated to provide an optimal environment
in which the 129/Sv ES cells can compete effectively with the ICM to produce
high quality chimeras. However, if investigators have access to
proven ES cells from another strain, the GEAM can work with them to identify
and obtain a suitable host strain assuming they are prepared to accept
any additional costs or risks that may be incurred.
the microinjections, the blastocysts are allowed to recover and transferred
into the oviducts of pseudopregnant recipients.
Pups should be born 19 days later and coat color can be identified
one week after that. Since the 129/Sv ES cells are from an agouti
strain, and the blastocysts are from a black strain, the more agouti that
is observed in a chimera, the better that founder is judged to be.
The founder chimeras will be ready to mate
to C57BL/6 partners at 6-7 weeks of age. Germline transmission will
result in the production of agouti F1s. Up to three chimeras from
each clone will be bred in the GEAM. Any additional chimeras will
be shipped to the investigator for breeding.
• Long-term storage/recovery
ES cell clones are stored in GEAM. A $80.00 yearly liquid nitrogen storage fee is charged per Principal Investigator in December regardless of the number of samples stored.
Investigators can request their ES cell clones at any time. ES cell clones will be processed according to Principal Investigator wishes.
ANIMAL AND EMBRYO SERVICES
Microinjection of CRISPR/Cas9 reagents into C57BL/6J embryos for the production of knock-out mice
Microinjection of CRISPR/Cas9 reagents into C57BL/6J embryos for the production of knock-in mice
Microinjection of transgenes into C57BL/6J embryos
Microinjection of purchased targeted ES cells into C57BL/6J blastocysts
Microinjection of CRISPR/Cas9 reagents into Sprague Dawley rat embryos for the production of knock-out rats
Microinjection of CRISPR/Cas9 reagents into Sprague Dawley rat embryos for the production of knock-in rats
Microinjection of transgenes into Sprague Dawley rat embryos