INVESTIGATOR REQUIREMENTS FOR TRANSGENIC PROJECTS
* A completed application
form and copy of your current ACUC approval letter can be submitted prior
to or along with your purified microinjection DNA. Provide ~ 5-10µg
linearized microinjection quality DNA in buffer supplied by the facility at
a concentration in excess of 20µg/ml along with a original picture of
an ethidium bromide stained gel showing 200ng of the transgene fragment and
appropriate molecular weight markers.
One of the most critical factors for the successful production of transgenic
animals is the preparation of DNA. High transformation rates can be achieved
when ultrapure DNA is used for microinjections. Purity of DNA is assessed
by the relative ease of the injections and the number of single-cell embryos
that continue to divide in culture following injection. Generally, 60-80%
of injected embryos will develop to the 2-cell stage. If 50% or more fail
to divide, the DNA is considered to be toxic and a new preparation will be
requested. The TTML recommended protocol
generally yields microinjection quality DNA.
* Breeding and subsequent experiments are the
responsibility of the investigator. Our staff will provide consultation on
animal handling, breeding and housing if necessary.
* Please provide reprints or list of publications
resulting from work completed by the facility.
TRANSGENIC SERVICES PROVIDED BY THE LABORATORY
* The DNA concentration will be verified by fluorimetry and tested for toxicity. If the DNA has an inadequate concentration or is toxic (i.e. less than 50% of the injected embryos develop in vitro overnight) it will be returned to the investigator. The initiation fee will be charged for this service even if the DNA is unacceptable.
* Currently, all transgenic mice are generated
by pronuclear injection. Tested DNA is microinjected into the pronucleus of
fertilized single-cell embryos. Injected embryos are then surgically transferred
into the oviduct of pseudopregnant females and pups are born 19 days later.
Microinjections will continue until approximately 30 offspring are obtained.
If there are no positives, a second set of injections (up to a total for 500
injected embryos per construct including both sets of injections) will be
performed. Consultation and Southern blot analysis (if PCR used for initial
transgenesis screen) is required, however, prior to a second round of injections.
If the investigator chooses not to continue injections, the project initiation
fee will be charged to cover the cost of DNA quantitation and toxicity testing
of the injection DNA.
If embryonic lethality is suspected after consultation, injections for analysis
of transient transgenesis could be performed.
FVB/N, an inbred derived from CD-1/ICR, and inbred B6SJL (C57BL/6 X SJL)F1
are routine strains available for microinjections. C57BL/6 mice are also available
at an additional cost.
* Tail samples from 19-day-old pups will be
sent to the investigator for analysis. If DNA is analyzed by Southern blot,
a picture of the hybridization results (a photocopy of the autorad is acceptable)
must be provided. Positive and negative controls, molecular weight markers,
restriction enzymes, transgenic and endogenous bands should be clearly indicated.
If screened by PCR, a picture of the gel is acceptable. In this case, please
label amplified transgenic band and clearly marked positive/negative controls
and molecular weight markers.
PCR analysis of genomic DNA for transgene integration is not routine but
is available on request and contingent on time considerations. Investigators
must supply primers and positive control templates.
* Upon positive identification, TTML will initiate
the transfer of the animals the investigator’s assigned mouse room.
Please note that a record of the investigator’s screening results must
be received by the facility before animals will be released. Mice should be
screened again (using a freshly obtained tail sample) as soon as possible
after their arrival to confirm initial results.
* For transient transgenesis projects, pronuclear injection will be performed in (C57BL/6 x SJL)F1 hybrid or FVB inbred zygotes to generate 5 pregnant females at the desired day of gestation.
Other services include:
* The facility will provide technical training
and consultation to investigators who wish to utilize these procedures in
their own labs.
* Educational programs, lectures and demonstrations are also provided by the facility.
* Redrivation from frozen embryos stocks
Blastocyst
Injections
Targeted Mutagenesis Project