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Basic biological scientists are often concerned with understanding the cascade of events that lead to specific biological end points, such as how cells are driven to enter cell division and the various proteins that control this process, or how the response to hypoxic conditions is regulated. Often this involves examining the ability of proteins to undergo multiple protein-protein interactions and subsequently determining which specific complexation events are key to a given cellular response. One of the most utilized approaches to dissect the key protein-protein interaction events utilizes the introduction of point mutation(s) into a cDNA that can express a mutated protein, this can lead to the disruption of a specific protein-protein interaction. An example can be found in the studies by Lamarche et al., who examined the ability of Rac and cdc42 mutants to interact with either Ser/Thr kinase p65 or Ser/Thr kinase p160 [45]. By introducing mutations in Rac or cdc42 that disrupted binding of a specific kinase, they were able to demonstrate which kinase cascades have to be activated to obtain a biological response such as G1 cell cycle progression.

The application of transgenic and knockout (null) mouse models to the study of how specific proteins regulate gene expression—which in turn modulates cellular homeostasis, toxicology, and carcinogenesis-has become relatively routine in the last decade. Advances made in the use of these model systems are based in large part on classic research undertaken in the 1970s and 1980s, when embryologists were examining the fate of cells within early embryos and attempting to introduce exogenous DNA into mammalian embryos. It was first demonstrated in 1974 that viral DNA could be detected in mice whose blastocele cavity was injected with SV40 DNA during preimplantation development [1]. It was later shown that Moloney murine leukemia virus could be stably incorporated into the germ line of adult mice after viral infection of preimplantation mouse embryos [2]. In 1980, it was reported that pronuclear injection of exogenous DNA could integrate and be detected in somatic tissue in mice [3].

In this chapter, the term “transgenic” mouse will be used for mice generated by microinjection of DNA into pronuclei of embryos. This type of model has numerous applications and takes advantage primarily of of expressing, or over-expressing, a gene product of interest in a mouse model (). In , the term mouse will be used for mice generated by microinjection of recombinant embryonic stem cells into blastocysts. This type of model also has numerous applications and focuses primarily on the phenotype observed in a mouse when a specific gene product is deleted, or silenced (). The term mouse will be used for mice that are generated by a combination of these Conditional null mice typically contain both a transgene that is usually used to regulate expression of Cre recombinase and a targeted allele containing flanking sites that enable deletion of the gene product in a tissue- and time-specific manner.

There are both “conventional” and “conditional” approaches to generating null mice, with the former approach being more common until the application of Cre/ and Flp/ systems were developed. Both of these approaches are similar, but conditional methods take advantage of transgenic mice that express recombinase. There are several very good text books describing the construction of null mice, which should be consulted for more in depth coverage of the advantages and disadvantages of this method [11,47]. The overall strategy is depicted in (). In order to generate a conventional null mouse, a DNA targeting vector must be designed using isogenic genomic DNA. Construction of complex targeting vectors has recently been greatly improved. Since the mouse genome is now sequenced, investigators can identify BAC clones containing the gene of interest without screening a genomic library. Additionally, a recently developed method, termed “recombineering”, allows for rapid construction in less than one month of targeting vectors containing selection cassettes and LoxP/FRT sites [48].

Cells and cell lines can be created from transgenic and null mice providing a way to differentiate between direct effects and those that are dependent on secondary changes in neighboring cells. Isolation of various cell types can be accomplished using a variety of methods that can be obtained from standard textbooks. Additionally, cell lines can be derived from cells obtained for this purpose. The application of cells and cell lines from transgenic and null mice is particularly well suited when embryo lethality occurs, assuming midgestationstage embryos can be obtained for collection of the cell type of interest. A number of cells have been used for these purposes, but for illustration the remaining portion of this section will describe the use of embryonic stem (ES) cells, embryonic fibroblasts (EF), and hepatocytes.