CHAPTER 31 Genetic Engineering and Biotechnology. The Techniques of Genetic Engineering Review of Principles Underlying Genetic Engineering.
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Genetic Engineering and Biotechnology
Biotechnology is the use of living organisms to carry out chemical processes for industrial or commercial application.
Successful genetic engineering depends not only on being able to carry out molecular cloning but also on knowledge of replication, transcription, translation, and the regulatory aspects that control all of these processes.
The choice of a cloning host depends on the final application. In many cases, the host can be a prokaryote, but in others it is essential that the host be a eukaryote (Figure 31.2).
Any host must be able to take up DNA, and there are a variety of techniques by which this can be accomplished, both natural and artificial. Figure 31.3 shows a nucleic acid gun for transfection of certain eukaryotic cells.
Special procedures are needed to detect the foreign gene in the cloning host (Figure 31.4).
If the gene is expressed, the presence of the foreign protein itself, as detected either by its activity or by reaction with specific antibodies, is evidence that the gene is present. However, if the gene is not expressed, its presence can be detected with a nucleic acid probe.
Shuttle vectors allow cloned DNA to be moved between unrelated organisms. A shuttle vector is a cloning vector that can stably replicate in two different organisms.
Many cloned genes are not expressed efficiently in a new host. Expression vectors have been developed for both prokaryotic and eukaryotic hosts.
These vectors contain genes that will increase the level of transcription of the cloned gene and make its transcription subject to specific regulation (Figures 31.5, 31.6). Signals to improve the efficiency of translation may also be present in the expression vector.
Reporter genes transcription of the cloned gene and make its transcription subject to specific regulation ( are incorporated into vectors because they encode proteins that are readily detected. These genes can be used to signal the presence or absence of a particular genetic element or its location. They can also be fused to other genes or to the promoter of other genes so that expression can be studied.
It is possible to achieve very high levels of expression of mammalian genes in prokaryotes. However, the expressed gene must be free of introns.
One can also use the amino acid sequence of a protein to design and synthesize an oligonucleotide probe that encodes it. This process is in effect reverse translation and is illustrated in Figure 31.9.
Many human proteins that were formerly extremely expensive to produce because they were found in human tissues only in small amounts can now be made in large amounts from the cloned gene in a suitable expression system (Table 31.1).
Many recombinant vaccines have been produced. These include live recombinant, vector, subunit, and DNA vaccines.
Genetic engineering can be used to develop transgenic organisms capable of producing proteins of pharmaceutical value.
Genetic engineering is being employed to make plants resistant to disease, to improve product quality, and to use crop plants as a source of recombinant proteins and even vaccines.