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 withes the TAF to provide. Please
contact Kathy Krentz (firstname.lastname@example.org),
(608) 890-3785 to schedule an initial meeting.
Gene Targeting Vectors
up an initial meeting with us as you are beginning to design your targeting
vector. We'd be happy to make recommendations, and may be able
to provide you with useful vectors or sequences.
a billing request for services provided.
technology is covered by patents and license agreements. UW investigators
should contact Mark Doremus, 262-0153 or email@example.com
to request addition to the Addendum of Authorized Users of Cre/loxP
for Vector Design
recommend incorporating the following features in your targeting vector.
- DNA from mouse strain
129/Sv to maximize homologous recombination frequency in 129/Sv ES cells.
If you choose to use ES cells from another mouse strain, the genomic
DNA used in your targeting vector must be from the same strain (isogenic).
The ES cells we routinely use are R1 ES cells
(Nagy et al., 1993).
However with sufficient time to make other arrangements, it may be possible
for us to obtain and use ES cells from another strain of mice.
- NEO as positive selection
and HSV-TK as negative selection
- five or more kb of genomic
DNA in the two homology arms, with a minimum arm size of 1kb
- a unique restriction site
that will be used to linearize the targeting vector at the junction
between one of the homology arms and the cloning vector
- mutations or loxP sites
should be placed as close to the positive selection cassette as possible
- We highly recommend using
Southern blotting to identify correctly recombined ES clones. Unique
probes and restriction enzyme sites that lie outside the homology regions
will be used in Southern blot screens to identify correctly recombined
NEOr ES clones.
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
- meet with us to review your targeting vector and strategy for screening
your ES clones to identify correctly targeted clones
- complete a billing
- obtain a MTA for R1 ES Cells
- initiate or obtain an Animal Use Approval or provide your Animal Welfare
- provide us with
an isolated bacterial colony containing your targeting vector and a
detailed restriction map of the targeting vector
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
- contact Kathy Krentz (firstname.lastname@example.org) to
discuss the timeline for your project. Clones are injected in
the order in which they are submitted, so it is important to contact
us ASAP to provide all required forms and
information. Clone must be amplified and re-genotyped prior to
submission (i.e. clones must be ready to go).
- complete a billing
- provide an IACUC approved animal protocol number
- provide evidence that
clones to be injected (or the lab that they were targeted in) are mycoplasma
the clone or clones to be injected. Clones may be provied in frozen
vials at least one week prior to injections or in culture on 35 mm plates
on the week of injection. It is recommended that clones be karyotyped
prior to submission for microinjection as this greatly increases your
chances for success.
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 TAF 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 TAF. Any additional chimeras will
be shipped to the investigator for breeding.
• Long-term storage/recovery
ES cell clones are stored in TAF. A $75.00 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.