in the genome to include a single DNA nucleotide change (mutation) or incorporating a genetic marker (such as a gene that produces a colored protein that can be seen with a microscope) is accomplished by a similar method and is often referred to as creating a "knock-in"—since a piece of DNA is inserted into a specific part of the genome.

The genetically modified ES cells can be used to create mouse chimeras, whose offspring will carry the genetic modifications into the ES cells. These mice can be identified by analysis of DNA made from a small tissue sample, usually taken from the mouse's tail. The genomic DNA can be analyzed by polymerase chain reaction (PCR) or Southern blot to identify the mice carrying the genetic change. These offspring will be heterozygous for the introduced mutation and can be interbred to generate a second generation, some of which will be homozygous for the introduced mutation. The homozygous mice will lack both copies of the normal gene—any differences to normal development displayed by the mice as a result of losing the gene of interest can be analyzed. This, in turn, can provide valuable clues to the function of the gene and its normal role in mouse physiology.

This experimental approach to genetic manipulation of mice, which was pioneered in the 1980s, has been used to modify thousands of mouse genes and has played a significant role in understanding many physiological and pathological processes. For example, mutations in the p53 gene show its involvement in cancer, and mutations in the psd-95 gene show involvement in learning and memory. Variations on these methods are also used to model human disease mutations in mice and to test drug therapies.

ES cells also have potential for treating human diseases through "cell-based" therapy. In this context, the ES cells (or their differentiated derivative cells) are transplanted into patients whose own cells are defective or degenerated. This strategy could have applications for diseases such as Parkinson's disease (which affects brain cells), diabetes (pancreatic cells) and heart disease (which affects heart muscle cells). An important clinical issue

Figure 2. A typical chimera. ES cell-derived tissue is identified as light patches in the mouse coat.

PCR polymerase chain reaction, used to amplify DNA

Southern blot a technique for separating DNA fragments by elec-trophoresis and then identifying a target fragment with a DNA probe heterozygous characterized by possession of two different forms (alleles) of a particular gene homozygous containing two identical copies of a particular gene il

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