EARTH AND LIFE SCIENCE

Introduction to Genetic Engineering

Genetic engineering has been widely used to manipulate protein

expression in plants and animals. The applications of genetic
engineering are wide and the potential of this technology is greatly
beneficial to humans. Genetic engineering has been used in the
propagation of insulin, in vaccines, in crops, and for many other
purposes. For this reason, it is important to understand the basics of
genetic engineering because it will become more prominent in the
years to come.

Defining Genetic Engineering

The availability of techniques and methods is where scientific progress
rests on. Over the past 35 years, advancements in science have been
demonstrated by genetic engineering. The field of genetic engineering
has grown so rapidly that in many laboratories all over the world, the
isolation of a DNA fragments from the genome of an organism is
widely practiced. The technology of genetic engineering is now widely
used in forensics, paternity disputes, medical diagnoses, genome
sequencing and mapping, and the biotechnology industry. Gene
manipulation is striking in that it can easily be done in a wide range of
laboratories and is accessible to many scientists.

The term genetic engineering is often thought to rather trivial or

emotive, yet it is probably the label that most people would recognize.
There are several other terms that can be used to describe the
technology, such as gene manipulation, gene cloning, recombinant
DNA technology, genetic modification and new genetics.

Although there are many complex and diverse techniques involved in

genetic enginerring, the basic principles are simple. The premise on
which the technology rests is that genetic information, encoded by
DNA and arranged in the form of genes, is a resource that can be
manipulated in various ways to achieve certain goals in both applied
and pure science. There are many ways in which genetic engineering
can be of value, including the following:

 Basic research on the structure and function of genes

 Production of useful proteins by novel methods

 Generation of transgenic plants and animals

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 Medical diagnoses and treatment

 Genome analysis by DNA sequencing

The mainstay of genetic manipulation is the ability to isolate a

single DNA sequence from the genome. This is the essence of gene
cloning and can be considered as a series of four steps. Successful
completion of these steps provides the genetic engineer with a
specific DNA sequence, which may then be used for a variety of
purposes. Gene cloning can be thought of as molecular agriculture,
enabling the production of large amouns of a particular DNA
sequence. The ability to isolate a particular gene sequence is a
major aspect of gene manipulation. The four steps to genetic
engineering are:

 Generation of DNA fragments

 Joining to a vector or carrier molecule

 Introduction into a host cell for amplification

 Selection of required sequence

One aspect of new genetics is that there are concerns being raised
about the applications of the technology. The term genethics has been
coined to mean the ethical problems that exist in modern genetics.
These concerns are also likely to increase in number and complexity as
genetic engineering ensues. The use of transgenic plants, gene therapy,
investigation of the human genome, and many other topics are of
concern. These concerns are not just concerns for the scientists, but for
the population as a whole. Recent developments, for instance, in
genetically modified foods have raised public backlash against the
technology. Additional developments in the cloning of organisms, and
in areas such as in vitro fertilization and xenotransplantation, raise
further questions.

The Process of Genetic Engineering

Many of the procedures used in genetic engineering can be carried
out in a basic laboratory. However, large-scale applications, such
as production-scale biotechnology, require major facilities and
investment. The requirements for genetic engeering can be
summarized as follows: general laboratory facilities, cell culture
and containment, and processing and analysis.

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General facilities include aspects such as laboratory layout and

furnishings, as well as the provision of essential services such as
water, electrical power, gas, compressed air, vacuum lines,
drainage, and so on.

Cell culture and containment are facilities that are important for
growing cell lines and organisms required for research. Most labs
have facilities for growing bacterial cells, with the need for
equipment such as autoclaves, incubators (static and rotary),
centrifuges, and protective cabinets in which manipulation can be
carried out. Mammalian cell culture requires more sophisticated
facilities. Plant and algal cultures require the use of lighting in
culture cabinets. In many cases, some form of physical
containment is required to prevent the escape of organisms during
manipulation. The overall type of containment depends on the
vector and host being used. Biological containment necessitates
that the host does not survive outside of containment.

The overall containment requirements will be specified by national

bodies that regulate gene manipulation, and these will apply to
both bacterial and mammalian culture facilities. Thus, cloning E.
coli may require simple facilities, whereas cloning using
mammalian cells may require more stringent safety requirements.

For the processing and analysis of cells and cellular components,

such as DNA, there are a variety of equipment that needs to be
used.

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Isolation of DNA and RNA

Every gene manipulation experiment requires a source of nucleic
acids, either in the form of RNA or DNA. It is therefore important
that reliable methods for isolating these genetic components be
available for genetic engineering. There are three basic requiments:
1) opening the cells in the sample to expose the nucleic acid for
further processing; 2) separation of the nucleic acid from other
cellular components; and 3) recovery of the nucleic acid in purified
form. Several techniques may be used, ranging from simple to
complex.

The first step in the isolation protocol is the disruption fo the

starting material. The starting material may be viral, bacterial,
plant, or animal. The method used to disrupt the starting material
should be as gentle as possible, preferably involving the enzymatic
degradation of the cell wall and the detergent lysis of cell
membranes. If rougher methods are used, the danger of shearing
large DNA molecules will arise.

Following cell disruption, most of the methods used in DNA and

RNA isolation involve a deproteinization stage. This can be
achieved by one or more extractions using phenol/chloroform or
phenol. Protein molecules partition into the phenol phase and
accumulate at the interface after the emulsification and
centrifugation phases. The nucleic acids remain mostly in the
upper aqueous phase and may be precipitated from the solution by
issuing ethanol or isopropanol.

If DNA preparation is needed, the enzyme ribonuclease (RNase)

can be used to digest the DNA in preparation. If mRNA is needed
for cDNA synthesis, further purification can be performed using
affinity chromatography. This type of chromatography uses the
oligo(dT)-cellulose to bind the poly(A) tails of the eukaryotic
mRNAs. This enables the removal of contaminants, which are
chiefly DNA, rRNA, and tRNA.

The technique of gradient centrifugation is usually used to prepare

the DNA, in particular, plasmid DNA (pDNA). In this technique, a
caesium chloride (CsCl) solution containing the DNA prepraration
is spun at high speeds using an ultracentrifuge. Over a long period,
which takes up to 48 hours, a density gradient is formed and the
pDNA forms a band at one position in the centrifuge ube. The
band may be taken off and the CsCl removed by dialysis, resulting
to a pure preparation of pDNA.

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Handling and Quantification of Nucleic Acids

During a cloning experiment, it is often necessary to use small
amounts of nucleic aicds. It is obviously impossible to handle these
amounts directly, because they are so small, so most of the
measurements that are done involve the use of aquaeous solutions
of DNA and RNA. The concentration of nucleic acids can be
determined by measuring the absorbance at 260 nm, using a
sprectrophotometer. An A260 of 1.0 is equivalent to a concentration
of 50 g ml-1 for double-stranded DNA or 40 g ml-1 for single
stranded DNA or RNA. If the A280 is determined, ratio indicates if
there are contaminants present, such as residual protein or phenol.
The A260/A280 ratio should be around 1.8 for pure DNA or 2.0 for
pure RNA preparations.

In addition to spectrophotometric methods, the concentration of

DNA may be estimated by monitoring the fluorescence of bound
ethidium bromide. This dye, which is toxic and carcinogenic, binds
between DNA bases (intercalates) and fluoresces orange when
illuminated with ultraviolet light. By comparing the fluorescence
of the samples with that of a series of standards, an estimate of the
concentration of nucleic acids may be obtained. This method can
detect as little is 1-5 ng of DNA and can be used when
spectrophotometric measurements are not possible due to
ultraviotet-absorbing contaminants. When the concentration of the

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solution has been determined, it is easy to dispense the liquid with

accuracy to obtain small amounts of nucleic acids.

In a variety of applications, the precipitation of nucleic acids is

essential. The two most commonly used precipitants are
isopropanol and ethanol, with the latter being preferred. When
added to a DNA solution in a ratio, by volume, of 2:1 in the
presence of 0.2 M salt, ethanol causes the nucleic acids to come
out of the solution. After precipitation, nucleic acids can be
recoved through centrifugation, which causes a pellet of nucleic
acid to form at the bottom of the test tude. The pellet can then be
dried and the nucleic acid resuspended in the buffer needed for the
next stage of the experiment.

Labelling of Nucleic Acids

A major problem in genetic engineering, and specifically in DNA
or RNA isolation, is that there is a need to keep track of small
amounts of nucleic acids. This can be done using tracers.

Radioactive tracers have been used extensively in biochemistry

and molecular biology for a long time, and procedures are now
well-established. The most common radioactive isotopes used are
tritium, carbon-14, sulphur-35, and phosphorous-32. Tritium and
carbon-14 are low-energy emitters, with sulphur-35 being medium-
energy emitter, and phosphorous-32 being a high-energy emitter.
Thus, phosphorous-32 is more hazardous than the other radioactive
isotopes and special care must be taken during its use. Due to the
hazards of working with radioactive dyes, alternative methods such
as enzyme-linked labels and fluorescent dyes have also been used.
However, radioactive tracing is still the preferred choice.
Radiolabelling is often used to describe the technique.

One way of tracing RNA and DNA samples is to label the nucleic
acids with a radioactive molecule (usually deoxynucleoside
triphosphate, labelled with tritium or phosphorous-32). This is
done so that portions of each reaction may be counted in a
scintillation counter to determine the amount of nucleic acid
present. This is usually done using calculations that involve taking
into account the radioactivity present in the sample.

Radiolabelling may also be used in the production of highly

reactive nucleic acids that are used for hybridization experiments.
These molecules are known as radioactive probes and have a large
number of uses. The difference between labelling for hybridization

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and for tracing purposes is that labelling for probes is largely one
of specific activity. That is, the measure of how radioactive the
whole molecule is. For tracing purposes, the activity is not as
specific and high specificity is not needed.

Gel Electrophoresis
To the genetic engineer, the technique of gel electrophoresis is
vital. It represents a way through which nucleic acids may be
visualized directly. The method relies on the fact that nucleic acids
are polyanionic at neutral pH; that is, they carry multiple negative
charges because of the presence of the phosphate groups on the
phosphodiester backbone of nucleic acid strands. Thus, the
molecules will migate towards the positive pole of an electrode
when placed in an electric field. The mobility of the DNA
fragments depends on the fragment length. The technique is carried
out using a gel matrix, which separates the nucleic acid according
to their size.

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The type of matrix used for electrophoresis has important for the
degree of separation achieved. This is dependent on the porosity of
the matrix. Two types of gel are commonly used: agarose and
polyacrylamide. Agarose is extracted from seaweed and can be
purchased as a dry powder that is then melted in buffer at an
appropriate concentration, and the agarose sets to form a gel.
Agarose gel electrophoresis is usually run using the submerged
agarose gel electrophoresis technique (SAGE). Polyacrylamid gel
electrophoresis (PAGE) is sometimes used to separate small
nucleic acid molecules, in applications such as DNA sequencing.
The pore size of polyacrylamide gel is small.

Electrophoresis is carried out by placing the nucleic acids in the

gel and applying a potential difference across it. The potential
difference is maintained until a marker dye reaches the end of the
gel. The marker dye is usually bromophenol blue. The nucleic
acids are usually stained using ethidium bromide and visualized
under ultraviolet light. These nucleic acids show up as bands,
which can then be photographed. The data from gel electrophoresis
can be used to estimate the size of the fragments through
calibration. This technique is particularly useful in restriction
mapping.

DNA Sequencing
A central part of modern molecular biology is the ability to
determine the sequence of genes.

By definition, the determination of the sequence of a fragment of

DNA requires that bases are identified in a sequential manner. This
also enables the identification of processive bases. There are three
main requirements for this to be achieved:

 DNA fragments need to be prepared in a form suitable for

sequencing

 The technique used must achieve the aim of presenting each

base in turn in a form suitable for identification

 The detection method must permit rapid and accurate

identification of the bases

The preparation and generation of DNA fragments in fairly simple.

The fragments are often cloned sequences that are presented for
sequencing in a suitable vector. Much more difficult, however, is

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the identification of the position of the fragment within the

genome.

The sequencing protocol is more of a technical endeavor rather

than an experimental one. There are several variants of the
procedure, but the most widely used techniques are based on the
enzymatic method. Whatever the method, the desired result is to
generate a series of overlapping fragments that terminate at
different bases and differ in length by one nucleotide. This is
known as a set of nested fragments. Assuming that the technique
has generated a set of nested fragments, the detection step is the
final stage of the sequencing protocol. Usually, this involves the
separation of the fragments using a polyacrylamide gel.

There are two main methods for sequencing DNA. The first
method, developed by Allan Maxam and Walter Gilbert, uses
chemicals that cleave the DNA at certain positions, generating a s
set of fragments that differ by one nucleotide. The same result is

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also generated in the same way by the second method, developed

by Fred Sanger and Alan Coulson. This involves the enzymatic
synthesis of DNA strands that terminate in a modified nucleotide.
The analysis of fragments is the same for both methods, which
involves gel electrophoresis and autoradiography. The enzymatic
method has now completely replaced the chemical method.

In the Sanger-Coulson method, enzymes are used to sequence

DNA. A copy of the DNA to be sequenced as made by the Klenow
fragment of RNA polymerase. The template for this reaction is a
single-stranded DNA. A primer must be used to provide the 3’
terminus for DNA polymerase to begin synthesizing the copy. The
production of nested fragments is achieved by the incorporation of
a modified dNTP in each reaction. These dNTPs lack a hydroxyl
group at the 3’ position of deoxyribose, which is necessary for
chain elongation to proceed. Such modified dNTPs are known as
dideoxynucleoside triphosphates (ddNTPs). The four ddNTPs (A,
G, T, and C forms) are included in a series of four reactions, each
of which contains the normal dNTPs. The concentration of the
dideoxy form is such that it will be incorporated into the growing
DNA strand infrequently. Each reaction thus produces a set of
fragments terminated at a specific nucleotide, and the four
reactions thenn provide nested fragments. The DNA chain is then
labelled using a radioactive dNTP in the reaction mixture.

Electrophoresis and Reading of Sequences

The separation of DNA fragments created during sequencing
reactions is achieved by PAGE. A single gel system is usually used
for standard laboratory procedures. The gel usually contains 6-20%
polyacrylamide and 7 M urea. The latter acts as a denaturing agent
to reduce the effects of DNA secondary structure. This is important
because fragments are being separated that differ in length by only
one nucleotide. The gels are very tin (0.5 mm or less) and are run
at high-power settings. These settings cause them to heat up to 60-
70 C. This also helps to maintain the conditions necessary for
denaturing. After the gel has been run, it is removed from the
apparatus and may be dried onto a paper sheet to facilitate
handling. It is then exposed to x-ray film. The emissions from the
radioactive label sensitize the silver grains, which turn black when
the film is developed and fixed. The result is an autoradiograph.
The sequence is read from the smallest sequence upwards. Using
this method, sequences of up to several hundred bases may be read
from single gels. The sequence data are then compiled and studied
using a computer. The computer can perform analyses such as

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translation into amino acid sequences and identification of

restriction sites, regions of sequence homology, and other
structural characteristics, such as promoters and control regions.

Recombinant DNA
The production of recombinant DNA cannot be done directly.
Thus, a vector is used. A vector used is often a plasmid, which is a
small, circular piece of DNA found in bacterial cells. First,
plasmids are isolated. Using restriction enzymes, they are cut open
and the new gene or DNA fragment is inserted with the aid of
ligases. Thus, recombinant DNA is formed, which is the DNA
from different organisms joined in a single molecule.

Bacterial plasmid is the vector most commonly used. Plasmids

used in genetic engineering are said to be under relaxed control.
That is, their replication is totally independent of chromosomal
replication. These plasmids may be present in copies of 10-700 per
cell. The most popular plasmid is pUC18. However, bacterial
plasmids cannot accept DNA fragments that are longer than 5000
base pairs. Thus, they are restricted to cloning smaller DNA
fragments.

For lager DNA fragments, specially developed bacteriophage

lambda chromosome can incorporate up to 15-60 kilobases of
DNA fragments. A central 1/3 of its genome is normally not
required for phage infection and therefore can be replaced by
foreign DNA. The chimeric phase DNA can be introduced into the
host cells by infecting them with phages. On the other hand,

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cosmids are vectors that combined the features of both plasmids

and bacteriophages. It can accommodate DNA fragments up to 50
kilobase pairs long. Since cosmids have no phage DNA, they
reproduce as plasmids upon introduction into host cells by phage
infection. Yeast artificial chromosome (YAC) is a specially
constructed linear yeast chromosome that can incorporate DNA
strands of up to 1 million base pairs.

DNA Insertion
The simplest methods for the insertion of recombinant DNA into
cells are transformation and transfection. In the context of cloning
E. coli cells, transformation refers to the uptake of plasmid DNA,
and transfection refers to the process of uptake of phage.

In order for transformation of E. coli cells to occur, the host cells

must be made competent. This is achieved by soaking the cells in
an ice-cold solution of calcium chloride. Then, the cells are mixed
with plasmid DNA, incubating on ice for 20-30 minutes. This
enables the DNA to enter into the cells. The transformed cells are
usually then incubated in a nutrient broth at 37 C for 60-90 minutes

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to enable the plasmids to become established and to allow for the

phenotypic expression of their traits. The cells can then be plated
out onto selective media for propagation of the cells harboding the
plasmid DNA. Transformation is an inefficient process in that only
a small amount of competent cells become transformed. On the
other hand, transfection occurs using the same protocols, except
that plasmids are replaced by phage DNA. Once recombinant DNA
is present, the vector used will propagate, resulting in clones of the
recombinant DNA.

Plant Transformation
Genetic transformation, which can sometimes be hereditary, is a
change in the genome of an organism or a cell brought about by
the uptake of foreign DNA. A wide variety of gene transfer events
comprise transformation. Transformation can be characterized by
the stability of transformation, the subcellular component that has
been transformed, and whether the transferred DNA is integrated
in a stable manner into the host genome.

There are a number of definitions of transformation. Stable

transformation pertains to the stable maintenance of genes inside
the host organism. Transient expression, on the other hand, is when
the foreign DNA can be detected in the organism for the first few
day, and then ceases to be replicated. This type of expression is
typical for non-integrated, chromosomal DNA. Integrative
transformation is when the gene is covalently integrated into the
host cell and the gene is inherited by the offspring. Nuclear
transformation is when the gene is transferred into the nucleus of
the cell and is confirmed by cellular fractionation. Organellar
transformation is a transfer into the plastid or mitochondria f the
cell, as confirmed using cellular fractionation. Lastly, episomal
transformation is when viral genomes are introduced into the host
genome. It is also stable over several generations in most cases.

Whole plants can be regenerated from single events. Plant

transformation is based on two events: 1) successful introduction
of foreign DNA into host cells; and 2) subsequent development of
a complete plant derived from the transformed cells. In vitro
regeneration is the technique of developing plant organs or
plantlets that have been isolated from the mother plant and
cultivated using media in a laboratory. Regeneration occurs via
organogenesis (the initiation of adventitious roots from plant tissue
or cells), or embryogenesis (formation of plants from somatic cells
through a pathway resembling normal embryogenesis from the

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zygote. Both of these processes can be initiated either directly

(from merismatic cells) of after the formation of a callus (mass of
undiffertiated parenchymal cells introduced by wounding or
hormone treatment).

There is a large variety of gene delivery methods that scientists use

in order to transform plants. These methods range from being
simple, to complex, and to experimental.

Agrobacterium is a well-established transformation vector for

many dicots and several monocots. It is also a promising vector for
gymnosperms. In this technique, Ti or Ri-derived plasmic vectors
are used. Another technique is the direct transfer of DNA to
protoplasts, which is also a well-established technique with a wide
host range. The plasma membrane is permealized (made
permeable) by DNA chemical agents or electroporation.
Microprojectile bombardment is a widely used technique for
transforming plant cells by the introduction of DNA via coated
particles.

Micro-injection is an effective gene delivery method allowing the

visual DNA targeting to cell type and intracellular compartment.
Macro-injection is a simple approach that is used to deliver DNA
to floral tissue using a hypodermic needle. Impregnation with
whiskers, is the suspension of plant cells mixed with DNA and
micron-sized whiskers. Laser perforations is composed of transient
expression observed from cells targeted with a laser microbeam in
DNA solution. Impregnation of tissues is the transient and stable
expression from tissue bathed in DNA solution or infiltrated under
vacuum. Floral dip is used for transformation and expression
following the dipping of floral buds in DNA solution. Pollen tube
pathway, on the other hand, is the germ line transformation by
treating pollen or carpels with DNA and the procedure remains
controversial. Lastly, ultrasonication is a stable transformation
using explants in the presence of DNA.

The application of transformed plants is one of the hallmarks of

biotechnology and genetic engineering. The main focus of plant
transformation is to make them herbicide tolerant. In addition,
resistances to antibiotic stresses, such as drought, or improved
nutritional content are being investigated. Another application is
the production of medically valuable proteins or chemicals
(biopharmaceuticals), or the production of edible plants containing
vaccines. Gene stacking became popular in recent years. Gene

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stacking is the introduction and targeting of several traits in one

species.

Animal Transformation
The field of animal breeding today is influenced by the application
and development of biotechnology. The common goal of all efforts
in this field is genetic progress within a population. Genetic
progress is defined as the improvement of genetic resources and,
ultimately, the phenotype outcome. Genetic progress is influenced
by several factors: the accuracy of the candidates chosen for
breeding; the additive genetic variation within population;
selection intensity (the proportion of the population selected for
further breeding); and the generation interval (the age of breeding).
The first three factord need to be increased in order to increase
genetic progress. On the other hand, the last factor, which is
generational, needs to be decreased.

Techniques that are available for biotechnology vary, but they can
be divided into two groups. The first group includes all
technologies that interfere with reproduction efficiency (e.g.
artificial insemination, embryo transfer (ET), embryo sexing,
multiple ovulation, ova pick-up and cloning, among others. The
second group of application is based on the molecular
determination of genetic variability and the identification of
genetically valuable traits and characteristics. This includes the
identification and characterization of quantitative trait loci (QTL),
as well as the use of molecular markers for improved selection
process. Quantitative traits are phenotypic characteristics that show
a distribution of expression degree within a population (usually
expressed by a normal distribution) and that are based on the
interaction of at least two genes (known as polygenic inheritance).
An example of this is human skin color, which is determined by a
number of genes. A QTL is a DNA sequence that is related to a
certain quantitative trait. Knowledge of loci respoinsible for a
certain quantitative trait and underlying genes can help select
individuals for further breeding, or to start genetic engineering of
the selected trait.

Transgenic animals are animals that carry a specific and deliberate

modification of its genome, which is analogous to the transgenic
plant. To establish a transgenic animal, foreign DNA constructs
need to be introduced into the genome of the animal using
recombinant DNA technology. The DNA construct should be
stable enough that it can be passed on to the next generation.

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Heritability of genetic modification can be achieved by creating an

animal that carries the modification of in the genome of its germ
line. Thus, all offspring derived from this animal will be
completely transgenic as they will carry the foreign DNA in their
germ cell lines and somatic cells.

Transgenic animals can be created for a number of applications.

Examples are to gain an understanding of its genetic code and the
functions of genes. They can also be used to study gene control in
organisms and to build genetic disease models. They can improve
the production traits of animals and to produce new animals.

There are a number of ways to create transgenic animals. One of

these is micro-injection, which was discussed above. It is based on
the procedure of injecting foreign DNA into a fertilized zygote.
The construct integrates randomly into the host’s genome.
Subsequently, the zygote continues embryonic development. The
embryo is then transferred to foster mother and develops into a
transgenic animal.

Embryonic stem (ES) cell technology has also been developed,

partly to overcome the low-yield problem of micro-injection. ES
are derived from embryos at an early age in embryonic
development. Pluripotency is the ability of these cells to
differentiate into any of the cell types and tissues found in the adult
organism. This technique allows for gene targeting. In addition,
genes can be introduced, removed or replaced (knock-ins and
knock-outs).

Somatic cell nuclear transfer (SCNT) is the current method of

choice for producing transgenic animals. It is also known as
somatic cell cloning and initially gained importance for the
possibility of cloning animals in theoretically unlimited numbers.
It can also be used to produce transgenic animals, with genetic
manipulation as an added benefit. The insertion of a transgene
construct into a specific, pre-determined DNA site of the host
genome is known as gene targeting. The process of contructing of
the transgene is more complex that random gene insertion, as is the
case when micro-injection is used.

Artificial chromosome transfer is another technique that uses

artificial chromosomes. Artificial chromosomes posses a
centromere, telomere and origins of replication, which are
sequences that are responsible for their stable maintenance within

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the cell. Artificial chromosomes have the benefit of being able to

carry large fragments of DNA.

Sperm-mediated DNA transfer uses sperm as a vector to deliver

transgene DNA to the oocyte during the process of fertilization.

Viral-vector mediated DNA transfer can accomplish transgenesis

by using virus-derived vectors. These vectors are specifically
based on the retrovirus class of lentiviruses. Genes that are
essential for viral replication are deleted from the viral genome.
The capacity for integration of the viral genome into the host cell is
maintained. Parts of the vector that have been deleted can then be
occupied by the transgene of interest. Viruses that carry the
modified vector are then produced in vitro and subsequently
injected into the perivitelline space of the zygote. This results in
the infection of the zygote and the integration of the viral genome
into the host genome.

Applications for Transgenic Animals

Almost three decades ago, the first transgenic mice were produced.
Since then, a lot of techonologies have been developed for efficient
transgenesis. There are two main interests with regard to the
production of transanimals. The first is to improve intrinsic traits,
such as milk production and disease resistance. The second is the
production of animals that produce novel products, such as
proteins needed for medicine and pharmaceuticals.

Engineering transgenic animals usually focuses on improved meat

production, improved carcass quality, and enhanced milk
production. The main goal for transgenic animal development
concerning milk production are increased milk production higher
nutrient content, or milk containing novel substances. For insatce,
most milk proteins are caseins, and transgenic cattle have extra
copies of casein genes. This results in elevated casein levels in
proteins.

Experiments in cattle ar focused on the myostatin gene, which is a

negative regulator of muscle mass, resulting a high increase in
muscle mass in animals that have modified or deleted myostatin
genes.

Animal pharming is the term given to the use of animals for the
production of pharmaceutical products. The costs for producing
transgenic animals are high, but it is a worthwhile investment for

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the pharmaceutical company, which is a multibillion dollar

industry. Since the production of human proteins mostly cannot be
carried out in microorganisms, the production of
biopharmaceuticals in transgenic animal bioreactors is a feasible
alternative. Pharmaceutical proteins and other compounds can be
produced from a variety of body fluids, such as milk, urine, blood,
chicken egg whites, and so on. Nevertheless, milk is the preferred
medium due to its large production volume.

Transgenic animals may also produce humal polyclonal antibodies.

Antibodies are the fastest growing set of new biopharmaceuticals,
which are used for cancer therapy, autoimmune diseases,
transplantations, infections, and immune deficiencies. This
possibility is currently being investigated.

The introduction of genes into organisms may also increase disease

resistance. These genes can target specific areas of the host’s
immune system. Diseases that are being investigated include
bovine spongiform encephalopathy (BSE) and brucellosis.

Aside from those mentioned above, other applications for

biotechnology include the production of vaccines, the diagnosis
and cure of genetic diseases, DNA testing for paternity purposes,
and nutrition physiology. The latter refers to the use of specific
enzyme to modify foods and thus improve the nutrient availability
and uptake by the animal. Prebiotics (substances that simulate
microbial growth) and probiotics (live microorganisms) as
additives in feed can increase nutritional content of food.

Glossary
Autoclaves: used to sterilize equipment

dNTP: nucleoside triphosphate

Radioactive tracers: used to label nucleic acids

Videos and Resources

Coding Life: The Future of Genetic Engineering

Basics of Recombinant DNA Technology

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EARTH AND LIFE SCIENCE

Genetic Engineering Benefits: Applications in Medicine

DNA Cloning

Recombinant DNA Technology and Molecular Cloning

Gene Cloning & DNA Analysis: An Introduction

References
Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P.
V., & Jackson, R. B. (2011). Campbell biology (p. 379). Boston:
Pearson.

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