A genetically modified mouse or genetically engineered mouse model gemm is a mouse mus musculus that has had its genome altered through the use of genetic engineering techniques. Determine number of breeders required to produce a cohort of mice for experiments, based on breeding scheme and strain reproductive capabilities, and set up. Together with patientderived xenografts pdxs, gemms are the. The levels of growth hormone in the serum of some of the transgenic mice were several hundred times higher than in control mice. Most transgenic cores have many years of experiences in their fields and have published many knockout mouse models for transgenic mice.
After founder transgenic or targeted mice have been identified, 80100 mice. Transgenic mice protocol the objective of providing transgenic services is to generate transgenic mice for the primary investigators. The effects of injection sites and age of prefounder mouse was to be elucidated. Genotype pups by pcr to identify those carrying the transgene. Breeding, care and maintenance of mice case transgenic and targeting facility basic techniques for breeding transgenic mice. It is also possible to develop lines of transgenic mice carrying very large dna constructs 600 kb transected into es cells. Standard operating procedure for mouse colony breeding. Breeding of the bac transgene to a homozygous state carries an increased risk of generating an unwanted phenotype for two reasons.
The transgenic mouse facility will perform experiments to clean a mouse line from pathogen infections. An efficient strategy for generation of transgenic mice by. Transgenic mouse and rat breeding services cyagen us inc. It can be obtained by crossbreeding of a mouse strain expressing at least one a. This background is also advantageous if the transgenic line is crossed with induced mutants that are maintained as congenic strains on this. Transgenic females are mated oneonone with a male, and transgenic males can be mated with two or three females at a time. Random transgenic mice taconics custom transgenics program employs pronuclear microinjection technology to generate geneticallymodified founder animals. She quickly requested that the importexport coordinator at great eastern university import for her two breeding pairs of the knockout mice. Transgenic mouse methods and protocols melissa larson. Making a conditional knockout mouse requires nearly twice as much time and work to complete successfully. The most current information and methodologies for the generation of transgenic mice are found in this protocol. If you have a mosaic mouse, transmission may be much sparser than mendelian, and you may have to breed several litters to find your transgene. Individual investigators may choose to purchase wildtype mice for breeding at their own cost. In transgenic mouse methods and protocols, second edition, expert research explore current advances in the field through detailed laboratory protocols.
Genetic drift can alter strain phenotype over time. As you can imagine, breeding such mice can get a wee bit complicated. Crispr microinjection information from the tmm core. The jackson laboratory has a mouse breeding strategies manual. Custom transgenic mice service price cyagen us inc. Protocol used to create transgenic mice using crisprcas9. Methods and protocols is a valuable resource for both novice and expert researchers who are interested in learning more about this evolving field. More detailed descriptions of the complex breeding schemes, and the full description of the methods for vaginal cytology, are available in the list of references.
Paulie orourke was elated when a colleague at another university developed a knockout mouse that was important to orourkes research. Transgenic mice custom transgene expression taconic. The figure shows the process to design, clone and test shrnamirs for transgenic es cells and mouse production. Transgenic mice protocol the science of transgenic. The expression of a transgene may affect a strains viability. Amending a protocol to import and breed knockout mice. Subsequent use of these animals is also exempt providing the experimental protocol does not involve the use of recombinant dna. Mate mice when they are sexually mature 6 to 8 weeks old. Review of protocols for breeding colonies can be a challenge for the iacuc for. The facility provides a comprehensive set of technical services, has a fully operational construction and gene targeting service, and offers an annual course in mouse handling and breeding. Breeding strategies for maintaining colonies of laboratory. Standard operating procedure for mouse colony breeding center. The expected time for completion of each stage is indicated on the right. As the methods used to generate them continue to advance, more and more impressive research studies make use of these mouse models to help genetic researchers come up with new treatments and drugs.
Breeding strategies for maintaining colonies of laboratory mice. Transgenic mouse genetically engineered murine model. The transgenic mouse and embryonic stem cell facility provides investigators with genetically manipulated mice through transgenic technology or embryonic stem es cell manipulation. We recommend that transgenic founders or chimeras be mated to c57bl6 mice. When there is more than one adult female mouse in a cage with a litter or litters, there can be no more than a total of 14 pups. Implant injected eggs into surrogate mothers to obtain offspring. Mouse breeding performance optimizing breeding performance factors to consider to optimize the breeding performance of laboratory mice, observe the following practices. For most transgenic projects, the deliverables are f0 founder animals. The procedure will include superovulating, mating, and collecting fertilized onecell embryos. Impaired breeding performance or reduced fertility in genetically engineered mice gem can result in the loss of valuable research time and. The transgenicknockout core was established in 1993 to produce transgenic and knockout mice for usc norris cancer center investigators and to increase the use of mouse technology through education and consultation to the cancer research community. Periodic mating keeps eggs healthy, but making all those eggs takes a lot of work, so individual females should only be mated once per week. Transgenic mouse methods and protocols springerlink. Comprehensive and stateoftheart, transgenic mouse methods and protocols, second edition is the ideal guide for all researchers interested in the latest information about the production and analysis of transgenic and knockout mice.
However, mice bred that early generally produce small litters. Appendix b of the ilar guidelines for the care and use of mammals in neuroscience and behavioral research 2003 provides guidance for estimating the number of animals required to maintain a mouse. Pronuclear injection for the production of transgenic mice. It may be a plant, an animal or microbes such as fungi, viruses and bacteria.
Make a pedigree for each transgenic founder or embryonic stem cellmouse chimera. Zebrafish undergo sexual reproduction, so breeding requires both male and female fish. No more than three 3 adult mice may be in a single cage with a litter present. Breeding protocols university of california, davis. The giant mouse developed from a fertilized egg transformed with a recombinant dna molecule containing the structural gene for human growth hormone and a strong mouse gene promoter. Transgenic mice have been instrumental in dissecting the role of various neuronal proteins under both physiological and pathological conditions. So are the costs, mouse room space, and related resources needed. If the first litter includes offspring that carry the transgene or targeted gene, germline transmission has been demonstrated, and further breeding to. Currently majority of our customers request transgenic mice on the c57bl6n inbred background because the genome and phenotype of this mouse line is well known. University of michigan transgenic core guide to breeding transgenic and knockout mice. Experiment 2 was to detect the effect of the promoter for driving the expression of the transgene. Maintain breeder pairs of various ages by replacing a percentage of them monthly or weekly.
In general, gensat bac transgenic mouse lines should be utilized in the hemizygous state. In other words, a genotype developed by the process of genetic engineering is called transgenic plural transgenics. Pups are ready for weaning at 34 weeks of age, and reach sexual maturity at 6 weeks. Genetically modified mice are commonly used for research or as animal models of human diseases, and are also used for research on genes. Each new transgenic or targeted mouse must be mated to a normal mouse to determine whether the transgene or targeted gene is incorporated into germ cells. Breeding characteristics are strain and environmentdependent. Uci transgenic mouse facility tmf breeding strategies for maintaining colonies of laboratory mice a jackson laboratory resource manual. If the protocol does involve the use of recombinant dna then the research is covered under iiid4a. Creating transgenic mice using crisprcas9 genome editing. Preparation of dna for injection and development of a genotyping strategy. Cre lox breeding for beginners, part 1 jax mice and. For this purpose we use primers for mouse beta globin. Remember to include a negative control of water only and a positive control of known transgenic mouse dna or linearized plasmid dna diluted in mouse tail dna to the one copy level. Most times, the transgene is transmitted in a mendelian fashion.
In short, generating transgenic mice involves five basic steps. A pipeline for the generation of shrna transgenic mice. Replace breeders before their reproductive performance declines. Mice breeding protocol universidad central del caribe. Chapters provide a general introduction outlining how to deal with mice and how to generate transgenic mouse models, explore the generation of conditional and induced knockout and transgenic. Then, after germline transmission has occurred and the mice are bred to homozygosity, the floxed mice are bred to a tissuespecific cre transgenic mouse, and in the resulting offspring, the exon will be removed only in that tissue. Handling of mice, mouse strains, mouse breeding basics.
Transgenic mouse service transgenic mice what you need to know transgenic and knockin mice have existed for years. Estimating animal numbers guidelines for the care and. Cryopreserve your unique strains with the jackson laboratory and refresh inbred. Make a pedigree for each transgenic founder or embryonic stem cell mouse chimera. Such animals include a transgenic mouse strain expressing recombinant glutaminyl cyclase and a. Inject transgenic plasmid or bac into the pronucleus of fertilized eggs. Research mouse models that incorporate the crelox system typically involve generating double, triple, quadruple, or even ntuple is that really a word. Komp and eucomm breeding strategies recombinase gensat breeding recommendations.
Maintaining a colony of transgenic mice can be challenging. Simply put, the investigator constructs a transgene with a promoter and a structural gene for example a reporter gene such as lacz or a transcription factor. Remember to include an internal positive control to ensure that the dna sample is amplifiable. Transgenic mouse methods and protocols has essentially the same format as previous volumes of the series methods in molecular biology. Notice that only one generation of breeding is required hooray. With pronuclear microinjection, a predesigned gene or genes are injected and randomly integrated into the mouse or rat genome. Uc irvine university transgenic core has an excellent guide to mouse breeding and reproduction. For example, mating the transgenic strain in figure 1 to a strain that expresses cre recombinase in mammary tissue produces double transgenic offspring that express thekras oncogene only in the mammary glands figure 5. This is usually done to propagate mutant or transgenic lines, or because the trait. The investigator is responsible the mouse purchase. Estimating animal numbers guidelines for the care and use of. Mouse breeding performance breeding performance of laboratory mice can be affected by many factors, including the following.
They are open to modification and should not be construed as a comprehensive set of rules. Males have slimmer bodies with a slightly red hue, while females have bigger, silver bellies full of eggs. Transgenic outline university of michigan transgenic animal model core this is an outline of the steps necessary to obtain a transgenic mice. Transgene injection information transgenic mice are constructed by injecting cloned dna into fertilized mouse eggs. These pluripotent genetically modified es cells can then be used to make mice with deleted genes gene knockout or targeted mutagenesis of genes thought to be involved in the aging process. Given that the transgenes are randomly integrated into genome, and a small percentage of founder animals display some degree of mosaicism, there is a possibility that some f0 animals do not transmit the transgene to their offspring.
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