The ability to engineer the mouse genome has proven useful for a variety of applications in research, medicine and biotechnology. Genetically Engineered Mouse Models (GEMMs) mice have become powerful reagents for modeling genetic disorders, understanding embryonic development and evaluating therapeutics. These mice and the cell lines derived from them have also accelerated basic research by allowing scientists to assign functions to genes, dissect genetic pathways, and manipulate the cellular or biochemical properties of proteins.
History of mice as a model for human disease
Classic genetic analyses are performed by observing a phenotype, designing the necessary cross-pollinations or matings, and using the resulting population to perform statistically significant experiments to find the mutation and to understand the function of the altered gene. Inherited human diseases provide researchers with many phenotypes, and although the human is the mammal we are generally most interested in learning more about, it is also an animal we cannot use for genetic experiments for obvious ethical reasons. Mice naturally develop conditions that mimic human disease, such as cardiovascular disease, cancer and diabetes, and the inbred laboratory mouse has therefore been used as a model organism to study inherited human diseases for nearly a century. Lathrop and Loeb (1) published the effects of hormones on the development of tumors in mice in 1916, and the mouse has remained as a favorite model for human disease because it has a relatively low cost of maintenance and a generation time that measures only nine weeks.
Developments in molecular biology, stem cell biology and genomics over the last 30 years have allowed researchers to create custom-made mice through gene targeting in mouse embryonic stem cells. Site-directed mutagenesis in embryonic stem cells and the phenotypic characterization of the corresponding knockout and/or knockin mouse, allows researchers to study gene function as it relates to the entire organism. Now, certain diseases that afflict humans but normally do not strike mice, such as cystic fibrosis (2,3) and Alzheimer's (see 4 for review), can be induced by manipulating the mouse genome and environment.
ES cells and chimeric mice
Embryonic stem (ES) cells are pluripotent cell lines with the capacity of self-renewal and a broad differentiation plasticity. They are derived from pre-implantation embryos and can be propagated as a homogeneous, uncommitted cell population for an almost unlimited period of time without losing their pluripotency and their stable karyotype. Even after extensive genetic manipulation, mouse ES cells are able to reintegrate fully into viable embryos when injected into a host blastocyst or aggregated with a host morula. After these pre-implantation embryos are implanted into a surrogate mother, they develop into mosaic offspring known as chimeras. The tissues of chimeric mice are comprised of a mixture of cells that originated from both the host embryo and the ES cells. The contribution of each originating cell population is seen most visibly in the fur, which is generally striped black (from host cells) and brown (from ES cells). Healthy ES cells can give rise to descendants in all cell types, including functional gametes to produce more and more mice containing the desired genetic modification (5). If the proportion of ES cell descendents in the coat of the animal is high, the probability that ES cells are represented in gametes is also high, since ES cells mix thoroughly with host cells early in embryogenesis. Most researchers choose one of three types of ES cells for their targetings: 129, C57BL/6 and F1 hybrids which are 50% of each 129 and C57BL/6. For the first 20 years of targeted mouse modeling, the 129 ES cells were used almost exclusively. These ES cells give rise to brown coat color because they are Aw/Aw (dominant White-bellied Agouti), and the host cells give rise to black coat color because they are a/a (recessive non-agouti). The ES cells used most commonly are from the 129 strain of mice, while the host embryos are from the C57BL6 strain of mice. If the chimeras are bred to a/a non-agouti mice (for example C57BL6 or Black Swiss), then any brown offspring (Aw/a) must have arisen from ES cell-derived gametes, and 50% of the brown offspring are expected to carry the genetic modification.
For much more information on the different types of ES cells, their characteristics and coat colors, please see the Generation of Chimeras page.
Transgenic mice
One of the simplest ways to study gene function in a mouse is exogenous expression of a protein in some or all tissues. For this type of genetic modification, a new piece of DNA is introduced into the mouse genome. This piece of DNA includes the structural gene of interest, a strong mouse gene promoter and enhancer to allow the gene to be expressed and vector DNA to enable the transgene to be inserted into the mouse genome. Successful integration of this DNA results in the expression of the transgene addition to the wild type, basal protein levels in the mouse. Depending on the goal of the experiment, the transgenic mouse will exhibit over-expression of a non-mutated protein, expression of a dominant-negative form of a protein, or expression of a fluorescent-tagged protein. By definition, transgenesis is the introduction of DNA from one species into the genome of another species. Many of the first transgenic mice fit this description well as they were generated to study the overexpression of a human protein, often an oncogene (6). Currently, the phrase "transgenic mouse" generally refers to any mouse whose genome contains an inserted piece of DNA, originating from the mouse genome or from the genome of another species, and the term includes the standard transgenic mouse as well as a knockin or knockout mouse.
To generate a standard transgenic mouse, a bacterial or viral vector containing the transgene and any desired markers are injected into the pronucleus of a fertilized mouse egg. The DNA usually integrates into one or more loci during the first few cell divisions of preimplantation development. The number of copies of the transgenic fragment can vary from one to several hundred, arranged primarily in head-to-tail arrays, and the transgenic founder mice are mosaic for the presence of the transgene. Founders are very likely to have germ cells with the integrated transgene, and therefore will be able to vertically transmit the integrated gene, and all cells of the progeny transgenic mouse contain the transgene. This method is relatively quick, but includes the risk that the DNA may insert itself into a critical locus, causing an unexpected, detrimental genetic mutation. Alternatively, the transgene may insert into a locus that is subject to gene silencing. If the protein being expressed from the transgene causes toxicity, excessive overexpression from multiple insertions can be lethal to some tissues or even to the entire mouse. For these reasons, several independent lines mice containing the same transgene must be created and studied to ensure that any resulting phenotype is not due to toxic gene-dosing or to the mutations created at the site of transgene insertion.
A protocol for preparing DNA for a pronuclear injection to make transgenic mice is posted in the Methods section.
Glossary
Exogenous expression
Non-typical expression of a gene, usually due to a change in or replacement of the promoter of the gene. Can cause an expression level that is higher, lower or differently regulated for that cell type.
Exon
A portion of a gene that contains sequence that codes for the protein
Gamete
In mammals, an egg or a sperm
Gene Enhancer
A region of DNA that is separate from the Gene Promoter that also affects the transcription of the gene. Enhancers have been found within introns or even several kilobases from the 5' or 3' end of the gene.
Gene Promoter
A regulator region of DNA a short distance from the 5' end of a gene that acts as the binding site for RNA polymerase.
Integrase
An enzyme that catalyses the insertion or removal of a piece of DNA into/from a larger piece of DNA.
Intron
A non-coding sequence located between exons of a gene.
Karyotype
The chromosome complement of an individual or cell, as seen during mitotic metaphase. In mice, a normal karyotype has 40 sister chromatid pairs.
Locus/Loci
The specific, physical location(s) on a chromosome (or chromosomes).
Morula
The globular mass of cells formed by the cleavage of the fertilized egg in the first stages of its development.
Oncogene
A gene that contributes to the production of a cancer. Oncogenes are generally mutated forms of normal cellular genes.
PCR
Polymerase chain reaction- a method for amplifying specific DNA segments which exploits certain features of DNA replication.
Pluripotent
Able to develop and differentiate into any of a large number of cell types. For example, pluripotent cells can become skin, blood, nerves, sperm or muscle in the right conditions.
Southern Blot
Transfer of electrophorectically separated fragments of DNA from the gel to an absorbent sheet such as paper. This sheet is then immersed in a solution containing a labeled probe that will bind to a fragment of interest.
Vector
In cloning, the plasmid or phage chromosome used to carry the cloned DNA segment.
References
- Lathrop, A.E.C and L. Loeb. (1916). Further investigations on the origin of tumors in mice: On the part played by internal secretion in the spontaneous development of tumors. J Cancer Res. 1:1-19.
- Snouwaert, J. N. et al. (1992). An animal model of cystic fibrosis made by gene targeting. Science 257:1083-1088
- Dorin, J. R. et al. (1992). Cystic fibrosis in the mouse by targeted insertional mutagenesis. Nature 359:211-215.
- Masliah, E. and E. Rockenstein. (2000). Genetically altered transgenic models of Alzheimer's disease. J Neural Transm Suppl. 59:175-83.
- Thompson, S. et al. (1989). Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells. Cell. 56:313-21.
- Robertson, E. et al. (1986). Germ-line transmission of genes introduced into cultured pluripotent cells by retroviral vector. Nature. 323:445-8.
Resources
Books
Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition)
By Andras Nagy, Marina Gertsenstein, Kristina Vintersten & Richard Behringer. © 2003 764 pp. (ISBN 0-87969-591-9) Available from Cold Spring Harbor Laboratory.
Cre/ loxP Recombination System and Gene Targeting: Transgenesis Techniques, Principles and Protocols, Second Edition. Ralf Kühn & Raul M. Torres 2002. pps. 175-204 (ISBN 1-59259-178-7)
Reviews
Muller U. (1999) Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis. Mech Dev. 82:3-21.
Nagy A. (2000). Cre recombinase: the universal reagent for genome tailoring. Genesis. 26:99-109.
Links
Nagy Lab Website — information about ES cells and the definitive Cre mouse database
MGI — MGI is the international database resource for the laboratory mouse, providing integrated genetic, genomic, and biological data to facilitate the study of human health and disease. It organizes information on transgenic animals and targeted mutations generated and analyzed worldwide, with an emphasis on knockout mice.