Transgenic mouse production Traditional founder production
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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 Knock-Out Mouse Core assists core users by providing cutting edge technical services in a timely and cost effective manner. 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 Loren Field, Core Director [email protected] 274- 5085 Bill Carter, Core Manager [email protected] 278- 0163 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. 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. OVERVIEW RESOURCES RESEARCH CONTRIBUTION HIGHLIGHT LIST OF SERVICES CONTACT INFORMATION 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. QUALITY CONTROL AND ASSURANCES 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. IUPUI Transgenic and Knockout Core
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IUPUI Transgenic and Knockout Core. RESEARCH CONTRIBUTION HIGHLIGHT . LIST OF SERVICES. OVERVIEW. Transgenic mouse production Traditional founder production Transient transgenic production (F0 analysis) Knockout mouse production Embryonic stem cell transfection Blastocyst injection - PowerPoint PPT Presentation
Transcript of Transgenic mouse production Traditional founder production
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 Knock-Out Mouse Core assists core users by providing cutting edge technical services in a timely and cost effective manner.
Transgenic mouse productionTraditional founder productionTransient transgenic production (F0 analysis)
Knockout mouse productionEmbryonic stem cell transfectionBlastocyst injectionChimera to germline breedingSingle cell line blastocyst injection (gene trap cell lines)
Assisted reproduction servicesIn Vitro FertilizationEmbryo cryopreservation pilot studyEmbryo cryopreservationSperm cryopreservationEmbryo thaw and transferTetraploid-ES aggregationArchival storage of cryopreserved embryos and sperm
Loren Field, Core Director [email protected] 274-5085
Bill Carter, Core Manager [email protected] 278-0163
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.
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.
OVERVIEW
RESOURCES
RESEARCH CONTRIBUTION HIGHLIGHT LIST OF SERVICES
CONTACT INFORMATION
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.
QUALITY CONTROL AND ASSURANCES
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.
IUPUI Transgenic and Knockout Core
