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IUPUI Transgenic and Knockout Core
OVERVIEW
The ability to genetically modify the mouse genome provides a
powerful approach to study many areas of biomedical research.
Transgenic mice harbor genetic modifications that result in the
expression of a new, exogenous gene (i.e., a gain of function
modification). In contrast, knock-out mice harbor genetic
modifications that result in the inactivation or the modification of an
endogenous gene (i.e., a loss of function or change of function
modification).
To generate transgenic mice, recombinant DNA molecules known
as transgenes are injected into the nucleus of one-cell embryos. The
transgene integrates at random sites in the genome. The injected
embryos are then surgically implanted into foster mothers and allowed
to develop to term. Approximately 20% of the resulting mice will have
stably incorporated the transgene. Once integrated, the transgene
can typically be transmitted to subsequent generations in a Mendelian
fashion. By incorporating a tissue-specific promoter into the
transgene, expression of virtually any protein can be targeted to any
cell type.
To generate knock-out mice, recombinant DNA molecules known as
targeting vectors are introduced into embryonic stem (ES) cells. The
targeting vectors are designed to undergo homologous recombination
at a defined genetic locus such that the recombination event will result
in either the disruption or modification of the targeted gene. ES cells
harboring the desired targeting event are injected into the blastocoel
cavity of embryonic day 3.5 blastocysts, which are then surgically
implanted into a foster mother and allowed to develop to term. The
resulting mice are chimeric, comprised in part from the ES cells and in
part from the host blastocysts. Chimeras in which the ES cell
contributed to the germ line are able to pass the ES-derived genome
to subsequent generations, thereby allowing the Mendelian
transmission of the gene-targeting event.
The methodologies used to generate transgenic and knock-out
mice are labor-intense, require specialized expertise with cell culture
and animal husbandry, and require specialized and expensive
equipment. Our core is well versed with the requisite techniques for
generating these transgenic animals. To date the core has injected
more than 320 different transgene constructs (resulting in multiple
lineages per construct) and produced germ line knock-out animals for
over 100 gene targeting constructs. Thus, the Transgenic and KnockOut Mouse Core assists core users by providing cutting edge
technical services in a timely and cost effective manner.
RESEARCH CONTRIBUTION HIGHLIGHT
LIST OF SERVICES
Transgenic mouse production
Traditional founder production
Transient transgenic production (F0 analysis)
Knockout mouse production
Embryonic stem cell transfection
Blastocyst injection
Chimera to germline breeding
Single cell line blastocyst injection (gene trap cell lines)
Assisted reproduction services
In Vitro Fertilization
Embryo cryopreservation pilot study
Embryo cryopreservation
Sperm cryopreservation
Embryo thaw and transfer
Tetraploid-ES aggregation
Archival storage of cryopreserved embryos and sperm
Doxycycline (Dox)-inducible expression of HIV Tat in astrocytes results in
neuropathy. Mice carry a GFAP/Teton and a TRE/Tat transgene. After 7 days of Dox
treatment gross disruption of the cerebellum was apparent. Tat transgene expression
was seen only with Dox treatment (middle row, red signal). The treated mice failed to
thrive, and exhibited hunched gesture, tremor, ataxia, and slow cognitive and motor
movement, seizures, and premature death. See Kim, B. O., Y. Liu, Y. Ruan, Z. C. Xu, L.
Schantz and J. He; Am J Pathol. 162: 1693-1707.
QUALITY CONTROL AND ASSURANCES
For transgenics via pronuclear injection we expect that the user will
find between five and ten transgene positive founder mice. If the result is
two founders or fewer, we will reinject the construct at no additional
charge.
For ES cell transfections we expect at least three correctly targeted
cell lines per construct. If there are fewer, we retransfect at no additional
charge.
For embryo cryopreservation pilot study, we thaw and transfer
embryos for two litters of mice. Resulting pups are screened by the
investigator for correct genotype. Positive mice can then be used to
generate additional frozen stocks if desired.
Following these guidelines we have generated over 320 transgenic
strains and 100 gene targeted strains of mice.
CONTACT INFORMATION
RESOURCES
Physical resources for the core include a dedicated, well
equipped cell culture laboratory with large redundant Dewars for
archival storage of ES cell lines, mouse embryos and mouse sperm.
Also is a dedicated embryo injection and mouse surgery laboratory
that includes three Leica injection stations, a satellite tissue culture
setup (for outside ES cell lines), and two programmable freezing units
for cryopreservation.
Other resources include a variety of cryopreserved transgenic
lines for in vivo recombination of conditionally targeted mice, an
experienced staff and a number of faculty members with a great deal
of experience in the design and production of transgenic mice.
Loren Field, Core Director [email protected] 274-5085
Bill Carter, Core Manager [email protected] 278-0163
A random insertional mutation produces spontaneous epithelial thymomas. A
gross view (panel A) and survey section (panel B) of the heart and thymus revealed
the presence of a multi-lobed thymoma tumor at the base of the heart. High power
views of H&E staining (panel C) and immuno fluorescence staining (panel D; green
indicates keratin immune reactivity and red indicates leukocyte common antigen
immune reactivity); note the admixture of keratin positive epithelial cells (e) and
lymphocytes (l). See Nakajima, H., Nakajima, H.O., Soonpaa, M.H., Jing, S. and
Field, L.J.; Oncogene 19:32-38.