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A 'gene knockout' or 'knockout' is a mutation that inactivates a gene function. These mutations are very useful for classical genetic studies as well as for modern techniques including functional genomics. In the past, knockouts of bacterial genes were often made by transposon mutagenesis. In this case, laborious screens are required to find a knockout in the gene of interest. Knockouts of other organisms have traditionally been made by first using in vitro genetic engineering to modify genes contained on plasmids or bacterial artificial chromosomes (BACs) and later moving these modified constructs to the organism of interest by cell culture techniques. Other methods utilizing a combination of genetic engineering and in vivo homologous recombination were inefficient at best. Recombineering provides a new way to generate knockout mutations directly on the bacterial chromosome or to modify any plasmid or BAC in vivo as a prelude to making knockouts in other organisms. The constructs are designed to the base pair and are not dependent on suitable restriction sites. A drug cassette can be placed anywhere within a gene or the open reading frame of the gene can be replaced with the drug cassette. Either way, the desired construct is selected for.

Recombineering provides the ability to make rapid, precise, and inexpensive genetic alterations to any DNA sequence, either in the chromosome or cloned onto a vector that replicates in E. coli (or other recombineering-proficient bacteria), and to do so in a highly efficient manner. Complicated genetic constructs that are impossible to make with in vitro genetic engineering can be created in days with recombineering. Recombineering with single-strand DNA (ssDNA) can be used to create single or multiple clustered point mutations, small or large (up to 10kb) deletions, and small (10-20 base) insertions such as sequence tags. Using optimized conditions, point mutations can be made with such high frequencies that they can be found without selection. This technology excels at creating both directed and random mutations.

"Recombineering," in vivo genetic engineering with short DNA homologies, is changing how constructs are made. The methods are simple, precise, efficient, rapid, and inexpensive. Complicated genetic constructs that can be difficult or even impossible to make with in vitro genetic engineering can be created in days with recombineering. DNA molecules that are too large to manipulate with classical techniques are amenable to recombineering. This technology utilizes the phage lambda homologous recombination functions, proteins that can efficiently catalyze recombination between short homologies. Recombineering can be accomplished with linear PCR products or even single-stranded oligos. In this chapter we discuss methods of and ways to use recombineering.

New hypotheses for the induction of autoimmunity have emerged based upon the interaction of lymphoid cells with superantigens (SAgs)—a new class of immunostimulants that are produced by viruses, bacteria, and mycoplasmas. SAgs are believed to play a role in the human autoimmune diseases including rheumatoid arthritis (RA) by clonally expanding anti-self T cells and by triggering polyclonal B cell responses. Mycoplasma arthritidis mitogen (MAM)— superantigen produced by an organism—causes chronic proliferative arthritis in rodents, in vivo expansion of collagen-reactive murine Vβ6 and Vβ8 T-cell receptors (TCR), and polyclonal activation of B cells. Furthermore, cytokine responses in injected animals shift toward a pattern that would favor the development of a humoral autoimmune response. Consistent with these properties is the ability of MAM to trigger and accelerate the onset of collagen-induced arthritis (CIA) in mice suboptimally immunized with collagen and to cause rapid flares in arthritis in animals convalescing from a previous episode of CIA.