Technology Genetic - 1
During the past few weeks we have discussed some of the ways in which the
structure of DNA can be changed in individuals through mutation, through "natural"
DNA rearrangements that occur in cells, and through recombination and
independent assortment during meiosis and sexual reproduction. We also examined
some of the genetic technology tools used to detect mutations and to analyze DNA
for molecular medicine, forensic and other applications.
For thousands of years humans have used selective breeding in agriculture and in
horticulture to obtain and maintain desired inheritable traits in many species of
plants and animals. In this sense, we have been manipulating genes far longer than
we have even known what "genes" are. We have taken advantage of the
capabilities of many organisms to manufacture foods and beverages we like:
yogurt, beer and wine, and cheese are examples of natural "genetic technology"
which produces things we humans find useful. The streptomycin drugs are
bacterial derivatives. Penicillium mold was one of the first organisms deliberately
"mutated" to produce better strains of the penicillin drug.
In addition, humans have, by our very treatment of our surroundings and those
with whom we inhabit our surroundings, been responsible (deliberately and/or
unintentionally) for the extinction of hundreds of species and their unique gene
combinations.
However, the direct manipulation of DNA for purposes of obtaining specific genes
to alter the genome of a target organism to do something we humans determine is
desirable dates from the 1970's, when researchers discovered the restriction
enzymes in bacteria that could cut DNA at precise nucleotide sequences and splice
the ends together. Bacteria use their restriction enzymes to remove potential
pathogens (viruses) from their genome, but the technique, once discovered,
proved to be one that can apply to all DNA. The field of genetic technology was
born and increasingly shapes our lives.
Genetic technology DNA technology, recombinant DNA technology,
biotechnology and g enetic engineering are all terms used for the field of
molecular biology that involves the manipulations of organisms or their
components to effect changes in an organism's DNA in very precise and directed
ways, for research or for industrial or commercial applications.
Much of today's DNA research is based on refining natural methods of
recombination and taking advantage of methods that can introduce different DNA
into cells resulting in recombinant DNA in the target cell or organism. The DNA can
be from the same or from different organisms. Organisms that have been altered
by means rather than by selective breeding are said to be g enetically modified
organisms or GMOs. Keep in mind that all DNA is composed of the same four
nucleotides no matter the origin of the unique sequence, and traditional means of
selective breeding also produce recombinant offspring genetically "modified" from
their parents. T ransgenic recombinant DNA research involves using DNA from
different species.
Genetic Technology - 2
Genetic technology has been used for a variety of purposes. Some applications of
genetic technology include:
Genes that promote ripening and softening of tomatoes have been
suppressed so that tomatoes can stay on vines longer (to develop the proper
flavor) but not get soft. Firm tomatoes are necessary for transportation
purposes.
Carnations have been developed with a gene insensitive to ethylene, the plant
hormone that stimulates maturation of fruits and wilting of mature plant
parts. Such carnations may last for weeks when cut.
Genetic technology research is involved in the analysis of gene sequences, to
provide knowledge about individual DNA for a variety of medical, forensic and
pure research purposes. With the use of small nucleotide polymorphisms or
SNPs and microarrays (see later), we can see differential expression of
genes in different tissues and under different conditions.
Some genetic technology research involves altering existing DNA to promote
or prevent the expression of certain genes to determine gene function, to
identify potential disease alleles and to experiment with ways to treat or
restore function once a test organism has lost function through "knocking
out" gene expression by altering its DNA.
Bacteria genetically modified to synthesize these substances produce
dozens of needed molecules.
Bacteria have also been modified to do jobs, such as chemical or oil spill clean
up. Modified bacteria (and some plants) can be used to remove toxins from
soil.
The golden rice developed by a Swiss consortium is an example of transgenic
recombinant DNA. Golden rice has genes to synthesize -carotene, not
naturally produced in rice grains. As many as 40 million children suffer
vitamin A deficiency in the world, and many of those children have a ricebased diet. Vitamin A deficiency is a leading cause of blindness in children
and is not reversible. Researchers used genes from a bacterium, daffodils
and corn to incorporate -carotene synthesis capability in rice. Golden rice
distribution was initially withheld from the market for a number of political
reasons. Golden rice is still controversial, and its benefits are mixed despite
the worthy goal.
Genetic Technology - 3
Genetic Technology Tools and Techniques
We shall briefly review some of the techniques and tools used in genetic technology
introduced in our section on mutations and molecular medicine. We will then
address some applications of genetic technology in medicine, agriculture, forensics
and research.
As with much of our molecular genetics, bacteria play an important role. Many of
our techniques originated with work with bacteria and were designed to produce a
recombinant bacterium that would synthesize a needed gene product. Synthetic
insulin was one of the first products produced using recombinant DNA technology.
Before we begin our discussion of techniques, let's look at one example of the use
of genetic technology in which we use bacteria to synthesize a needed human gene
product: insulin. Human insulin became available in 1982 with the use of this
process.
Several steps involved with a "typical"
recombinant DNA process to synthesize
a needed gene product.
Identify and isolate the gene(s) of
interest
Use restriction enzymes to
fragment and isolate the gene(s) of
interest
Choose the vector(s) that will
carry the gene(s) and that can be
cut by the same restriction
enzyme (s) for gene insertion.
Insert the gene(s) into the
vector(s) producing recombinant
DNA in the vector(s).
Insert the vector(s) into host cells.
Clone the host cells. (Not all cells
incorporate the vector(s).)
Screen the clones to find the gene
of interest. Vectors have
"markers" or reporter genes to
help identify successful host
incorporation.
Produce gene product in host.
Using Bacteria to Make Insulin
Genetic Technology - 4
The potential for recombinant DNA technology was demonstrated in 1973 when
Stanley Cohen and Herbert Boyer succeeded in cutting two antibiotic resistant
plasmids from E. coli with a restriction enzyme and splicing the two plasmids
together with DNA ligase, producing a plasmid resistant to both antibiotics. The
restriction enzymes could both excise a sequence of DNA and insert that sequence
into different DNA. They inserted the recombinant plasmid into E. coli cells lacking
the plasmids. The recombinant E. coli had enhanced antibiotic resistance.
To use this process in other research, locating genes of interest or otherwise
getting the DNA sequence of the gene, getting sufficient recombinant DNA to work
with, developing cloning methods, and finding vectors to carry the recombinant
DNA into target cells were all essential.
Finding the Gene of Interest
The desired DNA sequence or gene might be located and isolated by the following
methods:
Genome mapping facilitates identification of gene sequences.
Restriction enzymes make fragments of DNA more suitable for analysis.
Hundreds of restriction enzymes are now available for genetic technology
work.
Gel electrophoresis and Blots separate fragments.
DNA probes using RFLP and STR markers and DNA hybridization help locate
target gene sequences.
Once located, the target gene or DNA sequence must be made available in
quantities suitable for work.
The DNA polymerase Chain Reaction (PCR) amplifies the volume of the
desired DNA
Complementary DNA (cDNA), synthesized from RNA templates, can be used
when the DNA sequence can be determined.
Artificial DNA from known amino acid sequences is also synthesized.
In much recombinant DNA work, cloning libraries are developed of potentially
useful DNA sequences.
For transgenic or DNA recombination work, or for many DNA research
applications, promoter regions must also be combined with the target DNA
sequence.
Genetic Technology - 5
The gene or DNA must be transferred to the host, incorporated into the host's
DNA and expressed. In most cases this requires a vector.
Plasmids and viruses are often gene vectors.
Other techniques including the "DNA gun" and direct uptake of DNA into
target cells may be successful.
Successful recombination is determined by using reporter or marker genes
along with the target gene.
Some of these methods have been discussed in previous units. Other techniques,
such as complementary DNA, artificial DNA, vectors used and cloning libraries, will
be introduced here.
Complementary DNA (cDNA)
R everse transcriptase e nzymes
are used with a m RNA transcript
to code a complement DNA primer
strand.
The DNA strand can then be used
as a template to code its
complement to form an artificial
gene that works just like the
original DNA would.
The artificial DNA is called c DNA or
complementary DNA. An advantage
to this process is that we start
with a specific mRNA transcript
without introns.
Restriction enzyme recognition
sequences are added to the cDNA
and cDNA sequences are also
cloned for cDNA libraries.
Making cDNA from known mRNA transcript
Artificial DNA from Protein Structure
If the DNA sequence of the gene is not known but the amino acid sequence of the
protein we need is known, the DNA sequence can be determined by "working
backwards" to synthesize an artificial DNA molecule in the laboratory. The
sequences for transcription and translation signals in the host cell must also be
known and synthesized, as well as adding restriction enzyme recognition
sequences. Artificial DNA is useful for looking at the effects of mutations
because it is "easy" to make a mutated artificial DNA, often called a knockout
gene, which lacks the code for what one wants to find out. PCR can then create
the volume needed of the desired DNA sequence.
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The Vectors
Once isolated and a sufficient quantity of material accumulated, the recombinant
DNA gold is often to incorporate the desired gene or DNA into an existing genome
(host cell). This can be challenging since most cells do not readily take up "alien"
DNA to add to their genome, so a vector is used. The desired DNA can be
incorporated into the vector, and from the vector, into the target host. The host
may be a bacterium that can synthesize the desired molecule or make multiple
copies of a desired gene for basic research purposes. The host might be a plant or
animal or fungus. The techniques used for gene therapy, in which the gene of
interest is spliced into a vector for distribution in human cells, is similar.
DNA technology frequently uses modified p lasmids to add desired genes from
plasmid clones to host cells for research purposes, and for the manufacture of
chemicals needed by humans. The modified plasmids that carry DNA to the target
host are c loning vectors .
Two Common Plasmid Cloning Vectors
Plasmids are self-replicating and have restriction enzyme recognition sites, making
them ideal for recombinant DNA purposes. A plasmid typically will also have a
known g enetic marker , such as the antibiotic resistance of the R plasmids, or
plasmids with a nutritional deficiency, a fluorescent gene or other recognizable
feature, to facilitate locating a host that has incorporated a successful
recombinant plasmid. Such genes are often called r eporter genes.
Plasmid Vector with Fluorescent Reporter Gene
Plasmid Vectors with Antibiotic Resistance
Genetic Technology - 7
The plasmid may also have a s pecial prokaryotic promoter near the restriction
site where the gene will be added. This promoter will facilitate expression of the
gene if it gets successfully inserted into the bacterial host. Plasmids that have
these promoters are called e xpression vectors .
We can also make a rtificial bacterial chromosomes (BACs), each with a
centromere, telomeres and origin and the gene of interest. Libraries of BACs also
exist.
Yeast organisms can also be used as vectors because, unlike most eukaryotes,
they have plasmids. Yeasts are also unicellular and easy to grow. We also make
artificial yeast chromosomes called YACS. Using a eukaryote vector minimizes
problems that can arise with prokaryotic gene expression. A YAC must also have a
centromere, telomeres, an origin and the gene of interest. YACs also carry longer
DNA sequences than plasmids.
Viruses are often used as gene vectors for eukaryotes. Some of the possible
virus vectors are:
Retrovirus
Adenovirus
Adenoassociated Viruses
Liposomes
DNA
Genetic Technology - 8
For working with prokaryotes, b acteriophages are also used, and phage libraries
are almost as common as plasmid libraries.
There are also a variety of ways to directly transfer DNA into host eukaryotic
cells.
o DNA can be directly injected using fine needles.
o A technique called e lectroporation uses electric current to make the
plasma membrane temporarily more permeable to DNA by creating little
holes in the membrane.
o With plants, DNA can be attached to metal fragments and literally "shot" into
the plant using a DNA "gun" as well as plasmids (See later).
No matter what means we use to try and get a specific DNA sequence into our
target organism's cells, it is still chance whether the DNA will recombine in the
nucleus, be incorporated into the genome, and be expressed.
Creating and Storing Gene Clones in a "Genomic Library"
As mentioned in our overview, once cleaved by a restriction enzyme, the s ticky
ends of both the desired DNA and the cloning vector can be joined together by
DNA ligase, forming a recombinant DNA vector.
With bacteria, the successful host bacterium will pass the incorporated DNA on to
its descendants. This process is referred to as g ene c loning .
Because any given restriction enzyme will cut a number of DNA fragments, in
addition to the desired DNA sequence, thousands of different recombinant
plasmids or bacteriophages might be formed during the initial steps of recombinant
DNA work. A set of r ecombinant plasmids or b acteriophages will initially be
obtained to create a " DNA or genomic library" of genetic material for the
manufacture of desired substances. Subsequent steps may be needed to separate
out the recombinants that have the DNA sequence of interest from the rest of the
recombinants in the DNA library
Genetic Technology - 9
DNA libraries are important for genome mapping, but for single use purposes, we
need to isolate from a library the gene or DNA sequence with which we want to do
something.
We shall next explore ways in which the DNA information provided by the
techniques just discussed as well as other techniques are applied in many areas of
research and applications of genetic technology and gene expression.
Genetic Technology Applications - Studying Gene and Protein Function
The tools of molecular biology and genetic technology, as we have discussed in
previous sections of Biology 211 are applied in many fields, including medicine,
agriculture, horticulture, forensics and genetic analysis for disease prevention, as
well as for research into our evolutionary relationships and how life works in
general and in specific. We shall look at some of these applications now.
Inducing Mutations to Study Gene Effects
Just as Beadle and Tatum used mutant Neurospora to formulate their "one gene
one enzyme" hypothesis, researchers today continue to use mutations to study
not just what genes do, but to analyze how genetic information communicates how
protein products work. Mutations can be incorporated into artificial DNA
sequences can be recombined in target cells and the cells monitored to see the
effects. For example, the signal peptide sequences that target polypeptides to
the functional destination in cells was determined using artificial DNA with
deliberate mutations.
Genetic Technology - 10
Using Knockout Genes to Study the Effects of Mutation
In the late 1980s researchers first succeeded in deactivating a good gene in mice
so they could study the effects of a defective version of the gene. This has
proved useful in studying specific mutant alleles and developing effective
treatments, including restoration of the correct nucleotide sequences in the gene.
This process, known as in vitro mutagenesis, is often called genetic
knockouts, because the gene of interest has been "knocked out" or destroyed in
the test organism. We have many knockout mice today.
The technique uses common recombinant techniques with marker genes to ensure
that the mutated gene of interest has been inserted into the target embryo's
stem cells replacing the "normal gene" for analysis of the mutated gene's effect in
the mice.
Making a Knockout Mouse
Knockout mice were used to study a type of m ultiple sclerosis. There results
were impressive. Juvenile d iabetes in mice has also been successfully treated
with stem cell transplants for the islet cells that synthesize insulin after the
genes for insulin production were "knocked out". Mouse heart cells have been
cultured from embryonic stem cells, and have been successfully transplanted into
damaged heart tissue effecting repair. Huntington's disease, Alzheimer's and some
cancers have also been studied using knockout genes.
Transposons or insertion sequences are used in plants to create "knockout"
mutations and then the plants grown to observe affected phenotypes for the
traits being investigated. Knockout experiments have been done to identify
disease resistance and environmental stress resistance genes.
Genetic Technology - 11
Silencing Genes with Artificial RNA
Artificial RNA molecules, called antisense RNA can be synthesized that will block
(or interfere with) translation by binding to the real mRNA molecule thereby
preventing protein synthesis. In one cancer research, an antisense RNA that
blocked translation of a protein needed for cancer growth resulted in cells
reverting back to "normal".
Just as siRNA (interference RNA) can block translation by targeting the
degradation of mRNA as a normal mechanism of gene regulation, artificial siRNA
can also be made, which is more stable than antisense RNA. Both antisense RNA
and interference RNA are useful for identifying cause and effect relationships in
biological research. For example, the signal molecule that activates the events
associated with macular degeneration, a significant cause of loss of peripheral
vision as we age, was successfully blocked using an artificial siRNA.
Single Nucleotide Polymorphisms and Microarrays
Over 7 million single nucleotide polymorphisms (SNPs) have been mapped in human
genome analysis. SNPs have potential to provide each human with a DNA
identification for multiple purposes using a D NA microarray assay that results
in a gene microarray or DNA chip (or biochip), a small but discrete collection of
gene fragments on a chip.
In a microarray, several fluorescent single-stranded cDNA fragments are made
using mRNA and reverse transcriptase. The cDNA fragments are applied to the
DNA chip and where DNA hybridization occurs, the microchip fluoresces,
representing a gene that is expressed in the tissue sample.
With the use of DNA chips, researchers can compare an individual's s ingle
nucleotide polymorphisms ( SNPs) to the standard from the human genome
project, or to another DNA chip, identifying how the individual differs from the
standard. SNPs may be responsible for many genetic differences, from cystic
fibrosis and sickle cell anemia to red hair and cholesterol levels. A database of
hundreds of thousands of SNPs is being developed and may be a future way for
physicians to screen most accurately for many diseases.
Genetic Technology - 12
When a microarray was done on Arabidopsis, the genes coding for photosynthesis
enzymes were expressed in leaf tissue but not in root tissue.
SNPs and microarrays are also being used to personalize medical care. Since SNPs
are usually inherited as sets, called h aplotypes , within alleles they can be used
for disease identification and treatment (haplotyping) based on one's SNPs. About
50,000 haplotypes have been identified for this purpose.
There is some evidence that particular SNPs may correlate with increased risk of
some diseases. Certain mutations may result in a greater or lesser drug
response. Individuals may have or lack enzymes that negate or enhance a
particular medication. Such information may be able to provide data for better
health.
Genetic Technology - 13
DNA chip analysis can already be used in cancer screening to better target a
specific treatment to a specific cancer. Your DNA chip may also uniquely identify
all that is you, genetically speaking.
DNA Technology in Agriculture
Selective Gene Breeding
Although humans have been doing selective breeding for thousands of years, with
genetic technology, we no longer need several generations of breeding to get
desired characteristics, and even more importantly, we don't have to deal with
impacts of gene linkage that may bring negative traits along with the desired
traits. With some species, the young totipotent embryo can be "teased" apart so
that one zygote can be used to make a dozen identical offspring, each with the
desired genes.
Genetic Technology - 14
With specific gene selection, the insertion of the desired gene has to be at a gene
locus that can be "read" and must be inserted into gametes or at least into the
target tissue area to be useful. In addition, the inserted gene cannot disrupt
normal activity, and must have a recognizable promoter region for transcription in
the recipient's cells. Since there is little control over where the gene gets spliced
into the recipient's cells, mutant or transgenic organisms often have a low survival
rate.
Another advantage in specific gene selection is that the gene of interest can come
from any source; we no longer have to work within the genome of complement our
target organism, as in the development of golden rice some years ago.
Plant Applications of DNA Technology
Agrobacterium tumefaciens, a natural tumor-causing bacterium of plants, is the
host bacterium for much DNA technology in plants. The tumor genes are removed,
and a highly modified plasmid, the Ti plasmid, incorporates desired new genes,
along with the needed promoter, and post transcription processing genes. The
altered Agrobacterium "infects" a tissue-cultured plant (recall the ease with which
many plants can be grown from a single cell), which may express the inserted
genes normally. Hence we have many transgenic and genetically modified plants.
Embryonic Tissue
Transgenic cells (blue)
Transgenic Shoots
Successful Plants (left 2)
Transgenic Wheat Tissue Culture with Herbicide Resistance Gene
Use of the Agrobacterium tumefaciens Ti Plasmid for Gene Transfer
Genetic Technology - 15
Genes can also be "shot" directly into plant cells with a "DNA gene gun". The
gene gun injects coated DNA particles into the target plant cells.
Use of a DNA gun for gene transplant
DNA technology has been used to help plants become resistant to frost, saline
soils, wilting, herbicides such as round-up, insect pests and some fungal and virus
diseases. Genes have been altered to delay maturation and to alter nutrient
content. DNA technology research and accomplishments in plants include:
Herbicide resistance
A soil bacterium gene that is resistant to glyphosate, which blocks
chloroplast amino acid synthesis, has been introduced into a number of crop
plants, notably soybeans, using the Ti plasmid. RoundUp contains glyphosate.
Insect and other Pest Resistance
o The bacterium, Bacillus thuringiensis (BT), produces a protein that
forms a toxin in Lepidopteran larvae intestines, killing the larvae.
Desiccated BT has been used for years as a pesticide but degrades
rapidly once applied, so repeated applications are needed each year.
The genes that code for the BT protein have been incorporated into
cotton, corn, soybeans, tomatoes and potatoes, and also into strains
of bacteria (Pseudomonas) that invade root tissue.
o The enzyme, cholesterol oxidase, disrupts insect gut membrane
activity. The Bollgard gene that codes for the enzyme has been
isolated from a fungus, and has been used in potatoes and cotton in
field testing.
Environmental Tolerance
o Salt Tolerance
Some plants that grow in high saline soils have active genes that
transport excess Na+ into the vacuole. Genetic technology methods
have been used to activate that gene in plants normally not salt
tolerant.
o Frost Tolerance
A common bacterium, Pseudomonas syringae, which lives on stem and
leaf epidermis, causes ice crystals to form at temperatures above
freezing. This bacterium has been genetically altered so it cannot
make ice crystals so plant surfaces do not freeze until lower
temperatures are reached.
Genetic Technology - 16
Delayed Maturation
Tomatoes (the "Flavr Savr") that have delayed maturation can be harvested
and transported more readily, and have a longer shelf life.
Fungal Resistance
An altered bacterium helps elm trees resist Dutch elm disease.
Improved Nutritional Quality
As mentioned with golden rice, genes that enhance nutrient content can be
added to a crop plant.
o Enhancing protein quality is one goal by increasing the proportion of
amino acids that are using low in plants.
o Soybeans and rapeseed have had their fatty acids altered to improve
the quality of their oils to be "healthier" or to replace oils from other
plants currently used in foods that are more saturated.
Induced Sterility
Some genetically modified plants have been rendered sterile to prevent
interbreeding with wild varieties. This is to ensure that the genetically
altered genes do not spread into the "natural" environment.
Plants as Vaccine Vectors
It is hoped by some to introduce vaccines into fruits or vegetables, so that
obtaining immunity would be easier than it is today.
o Dutch researchers have incorporated a vaccine against dog parvovirus
into petunia, where the gene gets expressed in nectar producing cells.
Bees collect the nectar, and the vaccine is extracted from their honey,
demonstrating the feasibility of plants expressing the needed vaccine.
o Plants have also been used to produce human proteins, including milk
proteins used in hydration formulas.
Wilt Resistant Carnations
Weevil Resistant Peas
Round-Up Resistant Petunia
Salt Tolerant Plants
-carotene Rich Cauliflower
Slow Maturing Tomatoes
Genetic Technology - 17
DNA Technology for Improving the Environment
Bacteria are routinely used in sewage treatment and in composting wastes, both
necessary adjuncts to our human lifestyle. From the early research on splicing
genes into prokaryotes to degrade petroleum hydrocarbons, chlorinated
hydrocarbons and other toxic chemicals, researchers have found other ways to
take advantage of microbe capabilities. As do many plants, some microbes
selectively absorb certain minerals and metal ions. Such minerals can be
accumulated in bacteria for later recovery. With genetic technology, we have even
more possibilities for using organisms to repair our environment, each genetically
tailored for the specific job.
As is discussed in detail in Biology 213, plants can be used for b ioremediation of
polluted areas and as detectors of certain substances in soil because they take up
many substances in their roots. In one case, plants have been used in minefields
to locate mines left from previous military action. Often such plants have
incorporated marker genes to facilitate human recognition of their efforts.
DNA Technology Applications in Animals
Pharm Animals
Although the overall success rate in transgenic research on animals is low, virus
vectors or direct DNA injection can be used to incorporate desired DNA into in vitro
mammalian zygotes. Embryos are implanted into surrogate mothers. Successful
offspring develop into adults that produce certain needed human products. Such
engineered animals, with their "transgenes" are called pharm animals.
"Pharm" sheep milk contains a protein that minimizes lung damage associated with
respiratory diseases, including cystic fibrosis. Human blood protein genes inserted
into goat zygotes can be expressed in goat milk. Chickens have been used for
production of products that are expressed in their eggs.
Human growth hormone can be produced by bacteria in culture, but it is expensive
to do so. Cattle can produce human growth hormone in quantities in milk at a
reasonable cost making the needed hormone more widely available.
Testing must be done since the protein produced by the pharm animal may not be
identical in all respects to the more "naturally" produced molecule. Possible
allergic reaction to different proteins is a concern, as it is with genetically
modified foods.
Pharm Sheep
Pharm Goats and Milk Product
Genetic Technology - 18
Growth Hormones
Bovine somatotropic hormone (BST, also known as BGH) has been successfully
cloned and its use approved in the United States. BST increases milk production in
cows given the product, but at some metabolic and possible health cost. Cows
often have more mammary tissue infections, including inflammation. BST is also
being investigated to see if it increases muscle development in cattle and pigs.
We have a number of transgenic organisms into whose egg cells or early embryos a
growth hormone has been injected. Such animals reach maturity faster than
normal so that they can be marketed sooner.
Salmon given growth hormone
Mouse given human growth hormone
Other desired genes have also been introduced into animals in a similar fashion
resulting in a transgenic animal that expresses a trait more useful to humans.
Genetic Technology - 19
Other Genetic Technology Applications
Pharmaceutical Products
One of the earliest outcomes of DNA technology was the manufacturing of needed
molecules using the gene cloning techniques described earlier. E coli is used most
frequently as the recombinant organism, but some products are made from
incorporating the genes into yeast cells, or even mammal cells as just described
with pharm animals.
Formation of Tissue Plasminogen Activator
Molecules being manufactured today include:
Genetic Technology - 20
Medical Applications of Genetic Technology
As discussed earlier, molecular biology and DNA analysis have provided medical
research with the means to make great advances in the diagnosis and treatment
of many human disorders and diseases, including genetic disorders, cancers and
some pathogen-caused diseases. Genomics and proteomics add to the body of
information needed to tailor treatment to an individual's specific DNA functioning
using a combination of gene therapies, drugs, and preventive measures. Some
methods of identifying and treating genetic disorders were discussed in our
section on inheritance. Some Additional applications using genetic technology
research will be discussed here.
Stem Cell Research and Uses
One current research interest is stem cells, the cell lines that lead to the
development of precise tissue types, such as skin, immune system or blood cells.
At some point in development, stem cells are "programmed", do their job, and, as a
part of their programming, may even lead to programmed cell death, or apoptosis.
Such programming relies on genetic controls.
The cells of the early embryo are totipotent; all cells are undifferentiated. In
mammals, an early developmental stage called the blastocyst has stem cells that
are largely undifferentiated, or p luripotent . Such pluripotent embryonic stem
cells are particularly valuable for genetic uses. Stem cells are also found in
differentiated tissues, and are called t issue-specific stem cells or
multipotent stem cells. As tissues differentiate in development, some cells
remain, even in the adult, as stem cells, and have some use for those
differentiated tissue types.
Genetic Technology - 21
Because stem cells have more genetic "competence" they hold much promise in
gene therapy and for being host cells for gene implants, and pluripotent embryonic
stem cells more than multipotent cells. Embryonic stem cells have been used to
restore damaged and lost tissue in experimental animals.
Treating mouse embryonic stem cells with selected growth factors activates
the cells to differentiate into the tissues normally activated by those growth
factors.
Mouse heart cells have been cultured from embryonic stem cells, and have
been successfully transplanted into damaged heart tissue. There is promise
that the technique could work in humans, too.
Some human diseases that might be treated with stem cells are Parkinson's,
cystic fibrosis and diabetes. Stem cells can also be used to culture skin for burn
victims.
Despite the promise and reality of stem cell use in human health treatments, the
therapeutic cloning of embryonic stems cells for research and ultimately disease
treatment is an area of ethical concern for some since the sources for the stem
cells are donated embryos. Some countries, such as the United States, legislate
use of embryonic stem cells in research and use. Other countries do not do so
those countries have become leaders in stem cell research.
However, current research is showing that cells can be induced to act as stem
cells through genetic recombination. In Japan, Shinya Yamanaka at Kyoto
University, isolated genes that are abundant in embryonic stem cells and inserted
those genes into skin cells. The cultured skin cells expressed the stem cells' genes
and became functional pluripotent cells. They have demonstrated success in
diabetes, sickle cell anemia and in Parkinson's with their research animals.
Using Stem Cells to Produce Needed Tissue
Genetic Technology - 22
One common cancer therapy is the stem cell transplants (properly hematopoietic
stem cell transplantation or HSCP). Since chemotherapy kills rapidly dividing cells,
the chemicals also kill important blood and bone marrow stem cells. Prior to the
treatments, stem cells from blood and bone marrow and cultured in the laboratory.
After the therapy, the blood stem cells are reinjected. When successful, they
respond to growth factor signals and repair and replace needed cells.
Gene Therapy
Human gene therapy involves the transfer of human genes. When someone has a
non-functional or defective gene, a copy of the correct gene is transferred into
appropriate cells, typically using virus vectors, electroporation or direct DNA
injection. Stem cells that have incorporated a virus vector are frequent choices
for gene therapy. Alternatively, cells containing the normal alleles can be directly
transferred. Some diseases for which gene therapy may be possible include:
Cystic Fibrosis
Hemophilia
Muscular Dystrophy
Some Cancers
Familial Hypercholesterolemia
Rheumatoid arthritis
AIDS
Severe Combined Immune
Deficiency Syndrome
Gaucher Disease
Hunter Syndrome
Peripheral Vascular Disease
Gene therapy methods use gene cloning and virus vectors with bone marrow stem
cell transplants. The process involves:
Cloning a normal gene with DNA technology
Splicing the gene with a promoter region into a retrovirus that has
Implanting the virus into a bone marrow stem cell previously removed from
the affected individual
Introducing the "engineered" cells back into the bone marrow
The "new" marrow cells may code the introduced gene and make the needed
protein, and also reproduce themselves.
Genetic Technology - 23
Success varies with such gene transfer attempts. To be successful, a virus vector
has to be:
Accepted by the host cell
Insert its DNA in a codable location within the host cell chromosome
Must be in the appropriate tissues
Must be functional; that is make the substance the gene codes for in the
appropriate levels.
DNA insertion is random and can cause mutations in the host DNA depending on the
insertion location, or the viral DNA may cause problems in the chromosome and
negatively affect gene expression. In addition, the transplanted cells must not
trigger the immune system of the individual to reject them.
In 1990, two children were the first successful gene therapy recipients. Copies of
the DNA of their defective gene, ADA, were transplanted into isolated bone marrow
cells from each child. (ADA is a cause of severe combined immune deficiency
syndrome.) The modified cells were cultured and then re-introduced into the
children's bone marrow tissue. The transplant was successful and both children
survived, but other results were not as successful. Ten children were treated in
2000, but three developed leukemia. The cause was the insertion of the gene near
a gene that promoted WBC proliferation. Similar results occurred with mice that
developed lymphomas. Many questions have arisen including how to control the
insertion of the gene into the host cell DNA and prevent unintended consequences.
Human embryo stem cell
A gene therapy treatment that uses a genetically altered cold virus for the vector
was developed for cystic fibrosis treatment. The initial research was successful in
mice. The gene coding for the normal Cl- ion transport protein, the cf gene, was
spliced into an adenovirus and the virus sprayed into the lungs. The mice were
cured. However, the immune system of the mice used in the experiment was
disabled. When the experiment was tried in humans, after eight weeks, the
immune system cells attacked the altered lung cells, destroying them. And
adenovirus vectors often produce unintended secondary effects.
Genetic Technology - 24
Potential Medical Applications of Molecular Biology
Embryonic Correction of mutant alleles
Pre-implantation analysis today helps parents who are carriers of genetic
disorders select embryos for implantation that have the normal alleles. In the
future we might expect similar techniques to be used in conjunction with gene
corrections of "defective" genes. Embryos can be grown in culture with a vector
that carries the normal gene sequence. Genetically corrected nuclei can be
extracted from the embryo culture and implanted in enucleated eggs from the
mother. The genetically corrected egg can now be implanted and a "normal" child
can result.
Piggyback Vaccines
The vaccine for smallpox was based on the harmless cowpox virus, Vaccinia. There
is ongoing research to use this virus to create other vaccines, too. It is possible
to isolate the surface protein coat DNA (or RNA) from viruses such as H erpes
simplex and Hepatitis and splice the DNA into DNA extracted from Vaccinia. After
recombination, the altered Vaccinia DNA is reintroduced into the Vaccinia virus,
but the virus now carries protein markers for Herpes or Hepatitis on its surface.
Vaccinia injected into a human host will trigger the immune system to make
antibodies against the Herpes or Hepatitis protein, hence conferring vaccine
protection.
Genetic Technology - 25
Genetic Technology Ethics, Risks and Regulations
Some question the ethics of manipulating a genetic code that has evolved over
billions of years just because we humans have reached a stage in our knowledge
and technology when we can do so. Others are concerned about the risks of DNA
manipulation without knowing absolutely the consequences of doing so. (Oddly, we
seldom consider risk-taking in other human endeavors to this extent, but that is a discussion
for a different arena than Biology 211.) The following are intended only as questions to
initiate individual thought and consideration. They are not answers or
recommendations.
Much discussion surrounds genetically modifying foods. How much do we need or
want or have the right to know about genetically altered foods? Is it acceptable to
genetically alter foods the "traditional" way, but not to use current methods of
DNA technology? Are foods produced from organisms that have had gene
transplants (endogenous or transgenic) more likely to harm us than other things
routinely added to our foods? Is any altered DNA potentially hazardous? Is DNA
spliced into a food product mean that the DNA can now be incorporated into our
cells? Is the expression of DNA the same thing as a DNA transplant? Does
soybean oil contain any DNA at all, let alone altered DNA? These are questions all
of us must answer. Should we require any food that contains a product that
originated from a transgenic organism be labeled as genetically altered (a GMO)?
At this time we have no requirements that require labeling information for foods
that may be potentially contaminated with known harmful chemicals, but rely of
agencies such as the EPA, USDA and FDA to protect our foods from harmful
materials.
What happens if a gene splices into an inappropriate location in the target host's
DNA or has negative effects on the host organism? What do we do? Can we
destroy the GMO and revert back to the pre-GMO stock? The insertion of a gene
into a target has to be at the correct gene locus (with a promoter and terminator
location), and must be inserted into gametes or zygotes (or in tissue culture of
single cell isolates in plants) to be expressed in the whole organism, or at least into
the target somatic tissue area. In addition, the inserted gene cannot disrupt
normal activity. Mutant or t ransgenic animals often have a low survival rate.
Will the genetic alteration affect only the target organism and with the target
effect? Or might it affect some non-target organism deleteriously? For example,
using genetic engineering in plants to provide pesticide and herbicide resistance
means that we can grow crops with less use of the pesticides and herbicides that
pollute our air, water and soil. However, pollen from modified crops, particularly of
species that have wind pollination, spreads to other areas, affecting the gene pool
of the plant as well as impacting other organisms.
Genetic Technology - 26
Should we be concerned that, no matter how remote the likelihood, some altered
organism might escape and cause grave harm to our ecosystems? We have a
history of introducing organisms to new geographical areas with devastating
consequences to the native flora and/or fauna
In agriculture, there is hope of vastly improving plant productivity, by growing
plants in areas now poorly suited for agriculture, such as saline soils, or soils with
little moisture, or low fertility. What will this mean for the numbers of humans and
non-humans who inhabit this earth now? What impact will this have on the earth's
other resources? What about the impact on that area not now suited for human
agricultural practices? How much of the earth should we convert to agriculture
from its natural habitat?
When we succeed in getting animals to grow and mature much faster using growth
hormones, what impact does this have on environmental resources and the
carrying capacity? Salmon that mature five times faster do so by consuming five
times more resources in that shorter period of time.
With better screening ability, what are the ethical issues associated with screening
for life threatening diseases? Should we require genetic testing be accompanied
by appropriate education and counseling so that families can make informed
decisions about what they are discovering? There is always talk about insurance
companies or potential employers using genetic screening to screen out those
who might require costly treatments for their genetic diseases.
What about human cloning and making more copies of certain individuals? Or
choosing the genes one wants his/her children to have? What are the long-term
evolutionary consequences of reduced natural variability within the population?
How do we accommodate the beliefs of some that life begins at conception, and to
alter an egg or an embryo, or to destroy harvested eggs or embryos is destroying
a potential, if not realized life. Do we have the same sense of life with male
gametes? If not, why not?
No matter how we address these types of concerns, can we be assured that the
results of research that is conducted today under strict peer and national review,
and oversight commissions, will continue to be under such scrutiny in the future?
Moreover, rules developed in the past need to accommodate knowledge changes, so
rules and applications need the flexibility to change with more knowledge.
Each of us may be involved in making decisions about the use of genetic technology
for medicine, food production and forensics, the use of embryonic tissues in
research and treatment of diseases, gene experimentation on humans, and even
cloning. There are social, political and ethical issues that surround the science.
The more knowledge we have about molecular genetics, the better able each of us
will be to contribute intelligently to the decision-making processes.