Transcript Freeman 1e: How we got there

CHAPTER 31
Genetic Engineering and Biotechnology
The Techniques of Genetic
Engineering
Review of Principles
Underlying Genetic
Engineering
• 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 doublestranded 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).
• 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.
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).
• 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.
Finding the Right Clone
• 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.
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.
• 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.
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.
• 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).
Practical Applications of
Genetic Engineering
Production of Insulin: The
Beginnings of Commercial
Biotechnology
• 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.
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).
Genetically Engineered
Vaccines
• Many recombinant vaccines have been
produced. These include live recombinant,
vector, subunit, and DNA vaccines.
• Table 31.2 lists some genetically engineered vaccines.
• Figure 31.12 illustrates
production of recombinant
vaccinia virus and its use as a
recombinant vaccine.
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.
Genetic Engineering in Plant
Agriculture: Transgenic Plants
• 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).