A variety of strategies are used to manipulate the genomes of mice so that certain genes can be turned on, turned off, altered to work differently, to be monitored, or to be induced in particular life stages or locations. New strategies to accomplish manipulations and increase the precision of the methods are continually developed. The complexities of the experimental designs are beyond the scope of this site, but the general aspects of genetic modifications described below underlie all of these types of research paths. A treatise on engineering mouse genomes can provide additional details. The International Society for Transgenic Technologies also offers technical and professional resources in this arena.
N-ethyl-N-nitrosourea (ENU) mutagenesis
One way to generate modifications to genomic DNA is to use a chemical that damages DNA, and the resulting repair mechanisms sometimes result in errors in the sequence. N-ethyl-N-nitrosourea is a chemical known to promote this, and has been used in many types of animal screens to elicit genetically altered animals. An advantage of this method is that the randomness of the incidents means that preconceptions about mutations are not required. Sometimes referred to as “forward genetics”, this strategy means that researchers start with a phenotype of interest following the ENU screen and then look for the underlying genetic basis. New discoveries of unknown genes and mutations relevant to cancer may arise from this method. However, because of the availability of so many useful “reverse genetics” tools for mice that go from a gene to a phenotype to examine, more commonly researchers target mutations in genes expected to be involved in cancer using transgenic approaches described below.
To generate mouse models that carry novel, foreign, or modified genes and which are stably inherited, scientists have developed protocols that begin with preparation of the appropriate DNA segment, through injection or introduction of the prepared DNA into suitable recipient eggs or embryos, and ultimately the birth and characterization of the animals. An example of this process is illustrated on the right. Similar techniques are employed to create all types of transgenic mice: knock-out mice, in which a gene or segment has been removed; knock-in mice, in which a gene or segment has been newly added; or conditional transgenics, in which the altered components are under the control of some kind of regulatory switch to turn on or off the gene with various chemical, developmental stage, or tissue-specific mechanisms.
Preparation of the appropriate DNA construct requires careful planning to ensure that the ultimate product meets the research goals. Choice of appropriate promoter or regulatory switches may be a crucial decision: Should the novel gene be constitutively expressed (always on), or inducible? Should the gene be expressed in a given tissue or not? Does the gene carry some sort of tag that will enable localization or visualization of the product later? There may be size limitations that need to be met. Many aspects of the research need to be considered when planning the project, and will be unique to each investigation. Consideration of the mouse strain background is also important. Choosing appropriate inbred strains, or other engineered mice, is also crucial.
Direct Insertion of DNA
Insertion of the DNA can be accomplished in a various ways. It can be physically injected into prepared mouse pronuclei, or lentiviral vectors may be employed to deliver the DNA for integration into the genome.
Embryonic Stem Cells
Another way to accomplish genetic modification of mice is to employ embryonic stem (ES) cells to deliver the modified genomic content. The DNA construct is designed to have the desired characteristics, and then introduced to ES cells. The cells can be checked for appropriate integration of the DNA, and those cells can be injected into host blastocysts for transfer to foster mothers. Animals that stably carry and transmit the altered genomic content can be identified and bred.
RNA interference, or RNAi, is a technology to reduce expression of targeted genes using reverse-complementary sequences to interfere with gene expression. Short interfering RNAs (siRNAs) may be designed and introduced into mice to repress certain genes. For certain experimental designs this may be a faster route to explore the roles of genes or treatment situations.
Subsequent use of Genetically Engineered Mice
Whichever research path has been taken to obtain a modified mouse, these animals can be used as the primary research focus, or they can be bred with other mice to generate models of greater complexity. Maximizing mouse cancer models using the various possibilities of inbred mice, engineered mice, and even humanized mouse models enables tremendous strides in understanding cancer biology and translating our understanding to human health.
It is crucial to be aware of the rules of nomenclature for animal strains and for transgenic mice, so that the users of the models are able to understand the genetic background as fully as possible. Standards for designations of transgenic mice have been established to ensure that researchers can assess the alterations. Additional information about the nomenclature standards can be found in the section on Breeding Mice.