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
Much of genetic engineering is based on molecular cloning, in which a double-stranded DNA fragment from any source is recombined with a vector and introduced into a suitable host. Commonly employed cloning vectors include plasmids and bacteriophages.
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 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.
This can be accomplished by using reverse transcriptase to synthesize cDNA from the mature mRNA encoding the protein of interest (Figure 31.8).
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.
Fusion proteins are often used to stabilize or solubilize the cloned protein (Figure 31.10).
The first human protein made commercially using engineered bacteria was human insulin (Figure 31.11), but many other hormones and human proteins are now being produced. In addition, many recombinant vaccines have been produced.
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.
Figure 31.12 illustrates production of recombinant vaccinia virus and its use as a recombinant vaccine.
Genetic engineering can be used to develop transgenic organisms capable of producing proteins of pharmaceutical value.
One commonly used cloning vector for plants is the Ti plasmid of the bacterium Agrobacterium tumefaciens. The segment of the Ti plasmid DNA that is actually transferred to the plant is called T-DNA. This plasmid can transfer DNA into plant cells.
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.