Transgenic Animal Model Core

The Transgenic Core routinely prepares genetically modified mice and rats for University of Michigan investigators (transgenic mice, transgenic rats, knockout mice, and knockout rats). These animals can be used to study gene function, gene expression, gene regulation, to develop animal models of human disease, to test gene therapy reagents, to establish cell lines from specific cell types transformed in vivo, to produce mice with tissue-specific inducible gene expression or tissue-specific gene deletions, or to study the effects of cell specific ablation with toxigenes.

We provide access to our micromanipoulation and embryos stem cell workstations along with necessary reagents:

• specialized plasmids
• embryonic stem (ES) cell lines
• FBS
• feeder cells certified for ES cell culture.

The Transgenic Core provides assisted reporductive technology for mice and rats including:

• in vitro fertilization to exand animal colonies or to prepare 2-cell eggs for cryoperservation
• cryropresrvation of 8-cell eggs
• sperm cryopreservation
• recovery of live animals from cryopreserved materials
• intracytoplasmid sperm insertion (ICSI)

The Transgenic Core derives specific pathogen free mice or rats from pathogen infected animals.
Hands-on training is proved to individuals in all aspects of transgenic technology.

Refer to the list of services.

View the submission instructions.

The Transgenic Core prioritizes all requests for service on a “first-come, first-serve” basis. This standard is applied to everyone equally, even our faculty directors. Typically ES cell work is scheduled one to three months ahead. Since the inception of the Transgenic Core our policy has been that no project, whether it be gene targeting in ES cells or a transgenic mouse model, will enter the work queue until all of the required materials are provided. This includes both scientific materials, such as DNA samples and genotyping tests, and paperwork, such as approval to use animals in research, material transfer agreements and billing information.

Both transgenic and gene targeting efficiencies are excellent. We guarantee that at least three transgenic founders will be produced (the average number is 10). Since 1989 over 7000 transgenic founders were produced from over 700 transgene constructs as of September 2003. These efficiency of transgenic production equals or exceeds values in the published transgenic mouse  literature. The efficiency of all steps in gene targeting compare favorably with literature values. Multiple embryonic stem (ES) cell lines have been imported and screened for germline chimera formation. In addition, we have developed our own 129X1/SvJ ES cell line “Pat5”.  See the list of ES cell lines. We have collaborated with investigators to generate 70 new strains of mice (as of September 2003) from ES cells with mutations introduced by recombination with targeting vectors.

We provide advice on all aspects of this technology from experimental design to mouse breeding. We can provide protocols and training for every step in the process of generating transgenic or gene targeted mice. We are ready to interact, our doors are open, please contact us with any questions.

Any project that uses mice must be approved by the Institutional Animal Care & Use Committee (IACUC). Contact the Animal Care & Use Office for information on how to apply for permission to use vertebrate animals in research, testing, or teaching. The Unit for Laboratory Animal Medicine (ULAM) provides animal housing and veterinary care to all animals on campus. Investigators approved for animal research are expected to provide ULAM with a shortcode that can be used to pay for veterinary care and husbandry/housing costs. Transgenic Core submission forms require the following information:

• IACUC protocol approval number
• ULAM shortcode number for animal housing and veterinary care

Referencing The CCSG:

When referencing the Core Grant in any of your publications or other materials, please use the following citation:

This research was supported (in part) by the National Institutes of Health through the University of Michigan’s Cancer Center Support Grant (P30 CA046592).

Referencing Shared Resources:

Please remember our Cores!  When referencing any Cancer Center supported core in any of your publications or other materials, please use the following citation:

This research was supported (in part) by the National Institutes of Health through the University of Michigan’s Cancer Center Support Grant (P30 CA046592) by the use of the following Cancer Center Core(s):  xxx

A transgenic mouse or rat has a transgene in addition to its normal complement of genes. A transgene is an artificial gene cloned in the lab by recombinant DNA technology and microinjected into fertilized mouse or rat eggs. Eggs are transferred into foster mothers for gestation. Transgenic progeny are bred to produce a line. Transgenes integrate randomly into chromosomal DNA and are transmitted as a Mendelian trait.

The Core is available for consultation on all phases of transgenic research. The investigator designs and clones the transgene and develops a genotyping assay with single gene copy sensitivity, usually PCR based. A genotyping assay that detects an endogenous single copy gene in the mouse or an endogenous gene in the rat is a necessary positive control. The assays for the transgene and the endogenous gene are used to test all potential transgenic founder mice or rats. The combination of assays eliminates both false negatives and false positive mis-identifications. Transgene DNA is purified by the Transgenic Core for microinjection from a restriction digest supplied by the investigator. The Core microinjects DNA into fertilized (C57BL/6 X SJL)F2 eggs and transfers the eggs into pseudopregnant mice. Alternatively, we will make transgenic mice in other genetic backgrounds, upon request. We have successfully made transgenic mice in C57BL/6 X SJL)F2, FVB/N, C57BL/6,  (C57BL/6 X DBA/2)F2, SWR, B10D2 congenic strains, and mutant strains such as mnd2, Myo15sh2, and C57BL/10ScSn-Dmdmdx/J. When the pups are 2 weeks old, the Core applies ear tags and obtains tail biopsies from the mice. The Core will provide the tail biopsies to the investigator. Investigators may prepare genomic DNA from tail biopsies by hand or by any of the numerous kits on the market (see Protocols). The investigator will identify the transgenic mice by PCR. The transgenic mice are transferred to the investigator for breeding and analysis of transgene expression.

We guarantee that you will receive 3 or more transgenic mice or rats, however we can not guarantee transgene expression or transmission. The best strategy is to use a promoter that is already well characterize in transgenic mice or to employ very large flanking regions greater than 10 Kb. Alternatively, a bacterial artificial chromosome can be used to direct gene expression. BACs are include over 100Kb of genomic DNA sequence and often direct gene expression in a fashion which close matches the expression of endogenous genes.

The yield of transgenics is optimized by injecting highly purified linear DNA fragments with overhanging ends. Remove as much vector sequence as possible from the construct since prokaryotic sequences inhibit transgene expression. Although not a guarantee, demonstrated expression in a cell line is a positive indicator of in vivo expression and provides a rapid, inexpensive method to demonstrate that the transgene has been constructed properly. Contact [email protected] for more information on transgene design.

We guarantee that you will receive a minimum of three transgenic founders, often more. The purity of the microinjection DNA is the single most important factor which determines how many transgenic founders will be produced. Another very important parameter is the reliability and sensitivity of the screen for the transgene.

If you believe that your construct may be lethal, we will co-inject a neutral DNA fragment as a marker. If your transgene is not lethal then mice with both the transgene and neutral marker will be detected. However, if only mice with the neutral marker are detected then the construct is likely to be embryonic lethal. Alternatively you choose to examine transgenic founders. Transgenic embryos can be analyzed at various developmental ages to determine the time of death during gestation.

For the majority of transgenes the copy number does not correlate with expression level. The exceptions occur when large genomic fragments are used to make transgenics. Examples include P1 clones (90 Kb), BACs (180 Kb,) or YACs (400 Kb). Some reports suggest that if you use locus control regions or matrix attachment regions around your transgene that you may be able to insulate if form integration effects. Previous work in the literature has shown that attempts to produce low copy numbers by microinjecting dilute DNA does not affect copy number, but does reduce the overall yield of transgenic mice.

The size of your transgene should not interfere with transgenic mouse production. Large transgenes may be difficult to clone. You may wish to consider BAC recombineering to produce large transgenes under the control of regulatory elements in the BAC. The Core has produced transgenics from Bacterial Artificial Chromosomes up to 180 Kb in size. There are reports in the literature of transgenic mice produced from the microinjection of 90 Kb P1 clones, 248 Kb yeast artificial chromosomes, and even microdissected chromosome fragments.

Gene targeted mice are derived from embryonic stem (ES) cells. ES cells are manipulated in culture by introducing a targeting vector that is cloned in the lab by recombinant DNA technology. The targeting vector DNA precisely replaces a segment of chromosomal DNA (hence the name “gene targeting”) in the ES cell. ES cells are injected into a normal mouse blastocyst where they mingle with the embryo’ s cells to form the developing mouse. Up to 100% of the resulting mouse chimera can be formed from cells descended from the ES cells. ES cell-derived-mouse chimeras are bred to normal mice to produce progeny carrying the targeted gene which is transmitted as a Mendelian trait.

The major stumbling block in this process is the identification of ES cell clones that have undergone homologous recombination between the targeting vector and the chromosome in the ES cell. To maximize a successful outcome: 1) make sure that the genomic DNA in  your targeting vector is isogenic with the ES cell line question and 2) develop a hybridization screen that will detect your wild type gene in 2 ug of genomic ES cell DNA. We encourage you to contact [email protected] for a consult on your targeting vector design and on all phases of gene targeting research. The first step in the process is to obtain and map and sequence a 129X1/Sv genomic clone of the gene of interest. A 129X1/Sv library is available for screening from the core. The investigator designs and clones the targeting vector and purifies it for electroporation into embryonic stem (ES) cells. The Core has numerous plasmids designed for targeting vector construction. The targeting vector is then electroporated into ES cells by Core personnel, clones are picked after drug selection, the clones are cryopreserved at -80 C while the investigator screens DNA from the clones to identify those which have undergone homologous recombination. Alternatively, the Core will train you in ES cell culture and provide you with quality tested reagents and space in the multi-user Mouse Embryonic Stem Cell Laboratory so that you can do the work yourself. After the investigator isolates euploid ES clones which have undergone homologous recombination with the targeting vector they are microinjected into mouse blastocysts and transferred into pseudopregnant recipients. When the resulting pups are three weeks old, they are scored for ES cell contribution and transferred to the investigator for breeding and analysis.

• Characterize the genomic structure of your thoroughly.
• Develop a sensitive screen for homologous recombination in the ES cells.
• Consider the biological consequence of the mutation you introduce.

We can guarantee that we will inject your embryonic stem (ES) cells into a minimum of 50 blastocysts for ES cell-mouse chimera formation. Because of the intrinsic variability in individual ES cell clones, we can not guarantee that chimeras will be produced or that they will transmit your targeted gene through the germline. Therefore, we recommend that you provide at least three clones for microinjection. In collaboration with other labs on campus, we have successfully targeted over 60 genetic loci. We are confident in that we can work with you to genetically engineer new strains of mice that carry novel mutations of value to your research. The Core provides plasmids for gene targeting, ES cell lines that have been tested for germline chimera formation, feeder cells for ES cell culture, FBS tested for ES cell culture, and training in the exacting techniques required for successful ES cell culture. Contact [email protected] for more information on gene targeting projects.