I

Single-stranded brain cDNAs formed

Single-stranded cDNAs form template for double-stranded DNA I

Double-stranded brain cDNAs formed

Double-stranded cDNAs added to bacterial plasmids that insert the cDNA into the plasmid

Recombinant DNA plasmid containing the brain DNA

Recombinant DNA then inserted into bacteria which reproduce the brain DNA

cDNA library formed with each bacterium multiplying the specific cDNA it contains

Individual bacteria isolated and cultured to produce clones that yield specific cDNAs

Specific cDNAs (for example a receptor) inserted in plasmid that transfects a mammalian cell (e.g. fibroblasts) in culture I

Mammalian cell containing the specific cDNA then exposed in a culture medium to a toxin which destroys all non-transformed mammalian cells

Add radioligand that identifies the receptor of the mammalian cell I

Identification and isolation of transformed cells which can then be cultured to provide unlimited quantities of the receptor protein

Figure 5.1. Summary of principal methods used in molecular genetics.

subsequently isolated. Once a particular cDNA has been isolated in this way it can be used to make unlimited quantities of the macromolecule whose sequence it encodes. As mammalian cells are generally used for this method of amplification, the amino acid sequence is the same as that used in the limbic brain. Furthermore, if, for example, the cDNA encodes a neurotransmitter receptor, it is likely that it will be integrated into the plasma membrane of the cell surface and therefore largely reflect the portion of the receptor in the neuron. This enables such receptor-containing cells to be used for screening the affinity of putative psychotropic drugs on receptors that were derived from human brain. This method is now

commonly used in the pharmaceutical industry to screen numerous compounds for their potential therapeutic application: for example, screening compounds for their affinity for the human D4 receptor as potential atypical neuroleptics.

Another important application of cDNAs is to identify specific proteins in a tissue homogenate or tissue section. Since cDNAs undergo complementary base pairing, adding a radioactively labelled cDNA to a homogenate or tissue slice will bind it to the complementary sequence by a process of hybridization. Thus the amount of radioactive cDNA that hybridizes to the tissue or tissue extract is a measure of the amount of mRNA that is complementary to it. When this procedure is undertaken on slices of brain, it is known as in situ hybridization. In this way it is possible to determine the distribution of specific receptors in a tissue by accurately determining the distribution of mRNA that encodes for the receptor protein. This is a particularly valuable technique for the administration of psychotropic drugs.

A variety of techniques have now been developed to manipulate gene expression using cDNAs. For example, it is possible to introduce copies of a new gene (in the form of cDNAs) into a cultured cell line by a process of transfection. This is achieved by means of plasmids that transfect the human or mammalian cells in culture. Those cells that have had the DNA sequence integrated into their chromosomes can then be separated from those cells in which integration has not occurred by incubating the mixed cell population with a toxin to which the engineered cells are resistant whereas the normal cells are not. In this way clones of cells that contain the new genetic material can eventually be isolated.

A major advance in this technique has arisen through the development of transgenic mice. This technique involves injecting foreign DNA into the genome of the mouse embryo. As a consequence, the foreign DNA can give rise to a line of mice that contain the foreign DNA. Using this technique, mice have now been produced whose brains express the A4 protein-a marker for Alzheimer's disease. A variant of this technique is to replace a normal gene with a foreign gene in the chromosome, thereby giving rise to a progeny that lack both the normal gene and its function. Sibling mating then gives rise to offspring which have two defective genes. This method has so far largely been confined to mice which are termed ''knock-out'' mice. This method could prove to be particularly useful for determining the physiological role of specific neurotransmitter receptors.

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