Genetic Engineering and Biotechnology

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• CHAPTER 31
• Genetic Engineering and Biotechnology

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The Techniques of Genetic Engineering Review of
Principles Underlying Genetic Engineering
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• Biotechnology is the use of living organisms to
  carry out chemical processes for industrial or
  commercial application.

• 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.

• The techniques of genetic engineering are based
  on fundamental concepts in molecular genetics and
  biochemistry (Figure 31.1).

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• 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.

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Hosts for Cloning Vectors

• 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).

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• 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.

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Finding the Right Clone

• Special procedures are needed to detect the
  foreign gene in the cloning host (Figure 31.4).

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• 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.

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Specialized Vectors

• 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.

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• Many cloned genes are not expressed efficiently
  in a new host. Expression vectors have been
  developed for both prokaryotic and eukaryotic
  hosts.

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• 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.

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• 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.

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Expression of Mammalian Genes in Bacteria

• It is possible to achieve very high levels of
  expression of mammalian genes in prokaryotes.
  However, the expressed gene must be free of
  introns.

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• This can be accomplished by using reverse
  transcriptase to synthesize cDNA from the mature
  mRNA encoding the protein of interest (Figure
  31.8).

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• 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.

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• Fusion proteins are often used to stabilize or
  solubilize the cloned protein (Figure 31.10).

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Practical Applications of Genetic
EngineeringProduction of Insulin The
Beginnings of Commercial Biotechnology
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• 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.

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Other Mammalian Proteins and Products

• 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).

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Genetically Engineered Vaccines

• Many recombinant vaccines have been produced.
  These include live recombinant, vector, subunit,
  and DNA vaccines.

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• Table 31.2 lists some genetically engineered
  vaccines.

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• Figure 31.12 illustrates production of
  recombinant vaccinia virus and its use as a
  recombinant vaccine.

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Genetic Engineering in Animal and Human Genetics

• Genetic engineering can be used to develop
  transgenic organisms capable of producing
  proteins of pharmaceutical value.

• The techniques of genetic engineering are also
  applied to identifying individuals using DNA
  fingerprinting.

• One of the great hopes of genetic engineering is
  gene therapy, in which functional copies of a
  gene can be supplied to an individual to treat
  human genetic disease.

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Genetic Engineering in Plant Agriculture
Transgenic Plants
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• 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.

• 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.

• Commercial plants whose genomes have been
  modified using in vitro genetic techniques are
  called genetically modified organisms (GMOs).