A pipeline for the generation of shrna transgenic mice. Together with patientderived xenografts pdxs, gemms are the. Standard operating procedure for mouse colony breeding. Transgenic mice protocol the science of transgenic. Zebrafish undergo sexual reproduction, so breeding requires both male and female fish. It is also possible to develop lines of transgenic mice carrying very large dna constructs 600 kb transected into es cells. For this purpose we use primers for mouse beta globin. 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. In transgenic mouse methods and protocols, second edition, expert research explore current advances in the field through detailed laboratory protocols. 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. This is usually done to propagate mutant or transgenic lines, or because the trait. Transgenic mouse methods and protocols springerlink. This background is also advantageous if the transgenic line is crossed with induced mutants that are maintained as congenic strains on this.
Transgenic mouse genetically engineered murine model. Transgenic mouse and rat breeding services cyagen us inc. Random transgenic mice taconics custom transgenics program employs pronuclear microinjection technology to generate geneticallymodified founder animals. We recommend that transgenic founders or chimeras be mated to c57bl6 mice. Custom transgenic mice service price cyagen us inc. Genetic drift can alter strain phenotype over time. 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. Standard operating procedure for mouse colony breeding center. Mouse breeding performance breeding performance of laboratory mice can be affected by many factors, including the following.
As you can imagine, breeding such mice can get a wee bit complicated. 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. 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. It may be a plant, an animal or microbes such as fungi, viruses and bacteria. 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. Protocol used to create transgenic mice using crisprcas9. Cre lox breeding for beginners, part 1 jax mice and. 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. If the protocol does involve the use of recombinant dna then the research is covered under iiid4a. However, mice bred that early generally produce small litters. Transgene injection information transgenic mice are constructed by injecting cloned dna into fertilized mouse eggs. Experiment 1 was to optimize the protocol for generation of transgenic mice by lentiviral transduction of male germline stem cells. The effects of injection sites and age of prefounder mouse was to be elucidated. 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.
Transgenic mice custom transgene expression taconic. Males have slimmer bodies with a slightly red hue, while females have bigger, silver bellies full of eggs. In short, generating transgenic mice involves five basic steps. Genetically modified mice are commonly used for research or as animal models of human diseases, and are also used for research on genes. 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. Breeding, care and maintenance of mice case transgenic and targeting facility basic techniques for breeding transgenic mice. Transgenic females are mated oneonone with a male, and transgenic males can be mated with two or three females at a time. Breeding strategies for maintaining colonies of laboratory. After founder transgenic or targeted mice have been identified, 80100 mice. Notice that only one generation of breeding is required hooray. Transgenic mouse methods and protocols has essentially the same format as previous volumes of the series methods in molecular biology. 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.
In other words, a genotype developed by the process of genetic engineering is called transgenic plural transgenics. Subsequent use of these animals is also exempt providing the experimental protocol does not involve the use of recombinant dna. Uci transgenic mouse facility tmf breeding strategies for maintaining colonies of laboratory mice a jackson laboratory resource manual. The most current information and methodologies for the generation of transgenic mice are found in this protocol. Crispr microinjection information from the tmm core. Amending a protocol to import and breed knockout mice. Genotype pups by pcr to identify those carrying the transgene. Impaired breeding performance or reduced fertility in genetically engineered mice gem can result in the loss of valuable research time and. The transgenic mouse facility will perform experiments to clean a mouse line from pathogen infections. She quickly requested that the importexport coordinator at great eastern university import for her two breeding pairs of the knockout mice. Making a conditional knockout mouse requires nearly twice as much time and work to complete successfully. 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. Methods and protocols is a valuable resource for both novice and expert researchers who are interested in learning more about this evolving field. Most transgenic cores have many years of experiences in their fields and have published many knockout mouse models for transgenic mice.
The expression of a transgene may affect a strains viability. Breeding of the bac transgene to a homozygous state carries an increased risk of generating an unwanted phenotype for two reasons. 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. The jackson laboratory has a mouse breeding strategies manual. 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. Breeding strategies for maintaining colonies of laboratory mice. The levels of growth hormone in the serum of some of the transgenic mice were several hundred times higher than in control mice. The figure shows the process to design, clone and test shrnamirs for transgenic es cells and mouse production. Transgenic outline university of michigan transgenic animal model core this is an outline of the steps necessary to obtain a transgenic mice. The investigator is responsible the mouse purchase. Creating transgenic mice using crisprcas9 genome editing. Genetically engineered organisms are called transgenics. Make a pedigree for each transgenic founder or embryonic stem cellmouse chimera.
Estimating animal numbers guidelines for the care and. Transgenic mice have been instrumental in dissecting the role of various neuronal proteins under both physiological and pathological conditions. Handling of mice, mouse strains, mouse breeding basics. Transgenic mouse methods and protocols melissa larson. 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. Such animals include a transgenic mouse strain expressing recombinant glutaminyl cyclase and a. 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. The transgenic mouse and embryonic stem cell facility provides investigators with genetically manipulated mice through transgenic technology or embryonic stem es cell manipulation.
Maintain breeder pairs of various ages by replacing a percentage of them monthly or weekly. For most transgenic projects, the deliverables are f0 founder animals. Research mouse models that incorporate the crelox system typically involve generating double, triple, quadruple, or even ntuple is that really a word. Mice breeding protocol universidad central del caribe. 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. Breeding characteristics are strain and environmentdependent. With pronuclear microinjection, a predesigned gene or genes are injected and randomly integrated into the mouse or rat genome. Breeding protocols university of california, davis. No more than three 3 adult mice may be in a single cage with a litter present. Mate mice when they are sexually mature 6 to 8 weeks old. 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.
Pronuclear injection for the production of transgenic mice. Estimating animal numbers guidelines for the care and use of. This manuscript is an overview of the basic fundamentals of mouse and rat breeding and technical procedures used. Make a pedigree for each transgenic founder or embryonic stem cell mouse chimera. It can be obtained by crossbreeding of a mouse strain expressing at least one a. Remember to include an internal positive control to ensure that the dna sample is amplifiable. Cryopreserve your unique strains with the jackson laboratory and refresh inbred. Preparation of dna for injection and development of a genotyping strategy. An efficient strategy for generation of transgenic mice by. 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. Generation of genetically modified mice using crisprcas9 and haploid embryonic stem cell systems.
Paulie orourke was elated when a colleague at another university developed a knockout mouse that was important to orourkes research. 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. Uc irvine university transgenic core has an excellent guide to mouse breeding and reproduction. The crelox system can be used to turn off a transgene. Pups are ready for weaning at 34 weeks of age, and reach sexual maturity at 6 weeks. Most times, the transgene is transmitted in a mendelian fashion.
Implant injected eggs into surrogate mothers to obtain offspring. Maintaining a colony of transgenic mice can be challenging. Individual investigators may choose to purchase wildtype mice for breeding at their own cost. Review of protocols for breeding colonies can be a challenge for the iacuc for. Replace breeders before their reproductive performance declines. Transgenic mouse service transgenic mice what you need to know transgenic and knockin mice have existed for years. If the first litter includes offspring that carry the transgene or targeted gene, germline transmission has been demonstrated, and further breeding to. Mouse breeding performance optimizing breeding performance factors to consider to optimize the breeding performance of laboratory mice, observe the following practices. The expected time for completion of each stage is indicated on the right. Transgenic mice protocol the objective of providing transgenic services is to generate transgenic mice for the primary investigators. 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. The purchase or transfer of transgenic rodents requiring bl1 containment is exempt under appendix cvi.
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