When submitting
DNA for pronuclear injection, please use this DNA
purification protocol
Pronuclear
Injections for Transgenic Embryo/Strain Generation
The user
submits a Pronuclear Injection Request Form and provides DNA for microinjection,
purified using the core protocol. As the quality of the DNA is critical
for optimal success, extreme care must be taken in the preparation
of the DNA, and the core will request documentation of the purity and
quantitation of the purified sample. Microinjection of
DNA is performed routinely into FVB/N mouse embryos, although special
arrangements can be made for other strains, with the requesting investigator
covering additional mouse costs. Injected eggs are transferred
into a pseudopregnant recipient, and resultant pups are tagged and
tail biopsied for the detection of the transgene by the investigator.
Animals are housed within the core colony until genotyping is completed. The
founder animals are then transferred to the investigator.
Cost
for pronuclear injections for in-house MMCRI investigators is $400.00/injection
day. Two days of injections are recommended for each construct
to insure enough founders are produced. Approximately 100 eggs are
injected each day. In our experience, this leads to a minimum
to three to up to 20 founder lines generated.
*The
core facility is now accepting transgenic requests from investigators
outside MMCRI for $2,800.00 per construct, yielding a minimum of
three founder lines, assuming receipt of high quality DNA. Contact
Lucy Liaw to make arrangements (liawl@mmc.org).
Embryonic
Stem Cell Injections for Chimeric Mice
The investigator
submits an ES Cell Injection Request Form and provides ES cells that
have been tested and are negative for mycoplasma and mouse antibody
production (MAP) against murine viral pathogens. ES cells are injected
into C57BL/6 blastocysts. Each investigator should submit at
least 2 independent targeted clones to increase the chance of chimeric
mice being produced.
The cost
for ES cell injection for in-house MMCRI investigators is $800.00/clone.
Two days of injections are recommended, with approximately 50 blastocyst
injected.
Cryopreservation
of Morulae
The investigator
submits a Cryopreservation Request Form to initiate the service. The
number of mice needed for this service depends on many factors including
fertility of the strain, whether they are homozygous or heterozygous,
and genetic background. Core staff will assist in the planning of cryopreservation
of a line and determine the number of mice needed for the procedure.
The cost
for cryopeserving a mouse line for in-house MMCRI investigators is
$500.00/line. The cost to recover the line is $100.00.
Re-derivation
by Oviduct Transfer of Zygotes
The investigator
submits a Re-derivation Request Form. All mice that enter MMCRI
from non-approved vendors MUST be re-derived and serology tested before
housing within the barrier facility.
The cost
for re-derivation is $400.00/line. Core staff will assist in
determining requirement of mice for each line. The core will cover
the cost of importation and re-derivation of all shared lines.
