The cytokine TNF-α contributes to chronic inflammation in a range of conditions, including rheumatoid arthritis, by binding to and triggering signaling of the TNF receptor (TNFR). To treat these conditions, a fusion protein consisting of the constant-region domains of human IgG1 is fused to the extracellular portion of the TNFR, creating a therapeutic known as etanercept. When administered to patients, this fusion protein is effective at binding TNF-α and preventing it from triggering TNFR signaling, thereby reducing inflammation.
Fig. A.43 The function and expression of genes can be studied in vivo by using transgenic mice. DNA encoding a protein of interest, here the mouse MHC class II protein Eα, is purified and microinjected into the male pronuclei of fertilized eggs. The eggs are then implanted into pseudopregnant female mice. The resulting offspring are screened for the presence of the transgene in their cells, and positive mice are used as founders that transmit the transgene to their offspring, establishing a line of transgenic mice that carry one or more extra genes. The function of the Eα gene used here is tested by breeding the transgene into C57BL/6 mice that carry an inactivating mutation in their endogenous Eα gene.
A-34 Transgenic mice.
The function of genes has traditionally been studied by observing the effects of spontaneous mutations in whole organisms and, more recently, by analyzing the effects of targeted mutations in cultured cells. The advent of gene cloning and in vitro mutagenesis now makes it possible to produce specific mutations in whole animals. Mice with extra copies or altered copies of a gene in their genome can be generated by transgenesis, which is now a well-established procedure. To produce transgenic mice, a cloned gene is introduced into the mouse genome by microinjection into the male pronucleus of a fertilized egg, which is then implanted into the uterus of a pseudopregnant female mouse. In some of the eggs, the injected DNA becomes integrated randomly into the genome, giving rise to a mouse that has an extra genetic element of known structure, the transgene (Fig. A.43).
This technique allows one to study the impact of a newly discovered gene on development, to identify the regulatory regions of a gene required for its normal tissue-specific expression, to determine the effects of its overexpression or its expression in inappropriate tissues, and to find out the impact of mutations on gene function. Transgenic mice have been particularly useful in studying the role of T-cell and B-cell receptors in lymphocyte development, as described in Chapter 8, and in providing a source of primary T and B lymphocytes of known antigen specificity for adoptive transfer studies (see Section A-31). This utility is largely due to the fact that expression of the transgene-encoded T-cell and B-cell receptors preempts the rearrangement and expression of the endogenous antigen receptor genes during T-cell and B-cell development, respectively, thereby generating homogeneous populations of cells bearing a unique antigen receptor of known specificity.
A-35 Gene knockout by targeted disruption.
In many cases, the functions of a particular gene can be fully understood only if a mutant animal that does not express the gene can be obtained. Whereas genes used to be discovered through the identification of mutant phenotypes, it is now far more common to discover and isolate the normal gene and then determine its function by replacing it in vivo with a defective copy. This procedure is known as gene knockout, and it has been made possible by two developments: a powerful strategy to select for targeted mutation by homologous recombination, and the development of continuously growing lines of embryonic stem cells (ES cells). These are embryonic cells that, on implantation into a blastocyst, can give rise to all cell lineages in a chimeric mouse.