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Recombinant DNA is introduced into cells in tissue culture.

Recombinant defective retroviral DNA

Recombinant DNA is introduced into cells in tissue culture.

Reverse transcriptase and integrase

RNA copies of recombinant viruses are produced in cells containing a helper virus and packaged into viral particles.

Retroviral RNA genome with foreign gene

Retroviral genome with foreign gene is integrated into the target cell chromosome.

RNA copies of recombinant viruses are produced in cells containing a helper virus and packaged into viral particles.

Reverse transcriptase and integrase

Retroviral RNA genome with foreign gene

Retroviral genome with foreign gene is integrated into the target cell chromosome.

Recombinant virus particles infect a target cell.

Recombinant virus particles infect a target cell.

RNA packaged into viral particles. These particles can act as vectors to introduce the recombinant RNA into target cells. Viral reverse transcriptase and integrase enzymes (produced by the helper virus) are also packaged in the viral particle and introduced into the target cells. Once the engineered viral genome is inside a cell, these enzymes create a DNA copy of the recombinant viral RNA genome and integrate it into a host chromosome. The integrated recombinant DNA then becomes a permanent part of the target cell's chromosome and is replicated with the chromosome at every cell division. The cell itself is not endangered by the integrated viral DNA, because the recombinant virus lacks the genes needed to produce RNA copies of its genome and package them into new viral particles. The use of recombinant retro-viruses is often the best method for introducing DNA into large numbers of mammalian cells.

Each type of virus has different attributes, so several classes of animal viruses are being engineered as vectors to transform mammalian cells. Adenoviruses, for example, lack a mechanism for integrating DNA into a chromosome. Recombinant DNA introduced via an ade-noviral vector is therefore expressed for only a short time and then destroyed. This can be useful if the objective is transient expression of a gene.

Transformation of animal cells by any of the above techniques has its problems. Introduced DNA is generally integrated into chromosomes at random locations. Even when the foreign DNA contains a sequence similar to a sequence in a host chromosome, allowing targeting to that position, nonhomologous integrants still outnumber the targeted ones by several orders of magnitude. If these integration events disrupt essential genes, they can sometimes alter cellular functions (most cells are diploid or polyploid, however, so an integration usually leaves at least one unaffected copy of any given gene). A particularly poor outcome would involve an integration event that inadvertently activated a gene that stimulated cell division, potentially creating a cancer cell. Although such an event was once thought to be rare, recent trials suggest it is a significant hazard (Box 9-2). Finally, the site of an integration can determine the level of expression of the integrated gene, because integrants are not transcribed equally well everywhere in the genome.

Despite these challenges, the transformation of animal cells has been used extensively to study chromosome structure and the function, regulation, and expression of genes. The successful introduction of recombinant DNA into an animal can be illustrated by an experiment that permanently altered an easily observable inheritable physical trait. Microinjection of DNA into the nuclei of fertilized mouse eggs can produce efficient transformation (chromosomal integration). When the injected eggs are introduced into a female mouse and allowed to develop, the new gene is often expressed in some of the newborn mice. Those in which the germ line has been

FIGURE 9-33 Cloning in mice. The gene for human growth hormone was introduced into the genome of the mouse on the right. Expression of the gene resulted in the unusually large size of this mouse.

altered can be identified by testing their offspring. By careful breeding of these mice, researchers can establish a transgenic mouse line in which all the mice are homozygous for the new gene or genes. This technology was used to introduce into mice the gene for human growth hormone, under the control of an inducible promoter. When the mice were fed a diet that included the inducer, some of the mice that developed from injected embryos grew to an unusually large size (Fig. 9-33). Transgenic mice have now been produced with a wide range of genetic variations, including many relevant to human diseases and their control, pointing the way to human gene therapy (Box 9-2). A very similar approach is used to generate mice in which a particular gene has been inactivated ("knockout mice"), a way of establishing the function of the inactivated gene. ^ Creating a Transgenic Mouse

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TIt is difficult to summarize all the ways in which genomics and proteomics might affect the development of pharmaceutical agents, but a few examples illustrate the potential. Hypertension, congestive heart failure, hypercholesterolemia, and obesity are treated by pharmaceutical drugs that alter human physiology. Therapies are arrived at by identifying an enzyme or receptor involved in the process and discovering an inhibitor that interferes with its action. Proteomics will play an increasing role in identifying such potential drug targets. For example, the most potent vasoconstrictor known is the peptide hormone urotensin II. First discovered in fish spinal fluid, urotensin II is a small cyclic peptide, with 11 amino acid residues in humans and 12 or 13 in some other organisms. The vasoconstriction it induces can cause or exacerbate hypertension, congestive heart failure, and coronary artery disease. Some of the methods described in Section 9.3 for elucidating

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