Lecture 14 - Suraj @ LUMS

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Lecture 15

Biotechnology & Genetic
Engineering

Definitions

Biotechnology



the application of organisms/.

Biological systems/ processes in the manufacturing and.

Service industries
.


Genetic engineering



is a tool in biotechnology and.

Involves heritable, directed alteration of an organism’s.

DNA.



Aim


Make a living cell perform a
specific task in a predictable and
controllable way



Applications of Biotechnology


Environmental


waste management


Medical



low amounts of high value product



A) diagnosis



B) therapy



C) vaccine



D) research tools



E) human genome


Agricultural


large amount of low value product




More details in Chapter 12

Important Biotechonology
Products
-

Medical


Large scale production of penicillin.


Monoclonal antibodies.


Insulin was the first recombinant DNA
derived product approved for human use.


Human growth hormone was the second
recombinant DNA derived product
approved for human use.

Food and Drink Industry


Uses of yeast


Lactic acid fermentation


Single cell proteins


Bovine growth hormone


given to cows to
inc milk production. More production than
consumption. Gov. buys surplus


Service Industry
-
Enzyme Detergents


Dirt comes in many forms and includes proteins,
starches and lipids.


Using detergents in water at high temperatures and
with vigorous mixing, it is possible to remove
most types of dirt.


Cost of heating the water is high and lengthy
mixing or beating will shorten the life of clothing
and other materials.

Enzyme Detergents


The use of enzymes allows lower temperatures to
be employed and shorter periods of agitation are
needed.


At present only proteases and amylases are
commonly used. Although a wide range of lipases
is known, it is only very recently that lipases
suitable for use in detergent preparations have
been described.


Genetic Engineering


Why genes?


How can you change the genes of an
individual?


Examples of the use of this technology



Gene Vs Environment


DNA is the genetic material in all known forms of life.


DNA contains genes that give us many of our physical
characteristics.


The environment we are in also determines our traits.


One of the challenges of genetic engineering is to
determine how genes influence our traits and how to
modify DNA to alter these traits.


Genes affecting disorders such as alcoholism provide only
a predisposition. Having the gene for alcoholism may
make one more prone to alcoholism but does not guarantee
that one will become alcoholic, nor does not having the
gene mean one is immune.


Cells Used in Genetic Engineering


An important distinction in genetic engineering is between
germline

and
non
-
germline cells
.


Germline

cells are the eggs and sperm in humans.
Non
-
germline

cells are all the other cells in the body
-

muscle
cells, skin cells, liver, etc.


If a genetic modification does not alter germline cells, it
should not have any effect on the genetic makeup of future
generations.


If we were to introduce the gene for purple hair into mouse
hair cells, the
offspring

would
not

have purple hair, but the
parent

would.


If the gene for purple hair were introduced into the parental
germline cells, then the children
could

carry the purple hair
gene.


Dolly



No one thought that it would be possible to take a cell from an adult
mammal and use it to grow another, genetically identical clone. But
that is how Dolly was created.


She began life as a single cell taken from the udder of her mother.


The cell's nucleus was removed, transferred into an egg from which the
DNA had been removed, cultured and then implanted as an embryo
into the womb of a surrogate sheep.


Five months later, Dolly was born and is now a healthy lamb, a clone
of her mother
-
and without a biological father.


Dolly is very different from earlier sheep clones produced from cells
taken from embryos. Cells at such an early stage are undifferentiated
and it is not surprising that they can create all the different tissues of an
adult organism as they divide.


But cells from an adult were expected to be different
-
once an udder
cell, always an udder cell. That the biological clock can be wound back
and development run again as normal from the beginning is contrary to
biological dogma.


Now we know that we can clone an adult animal


Why Alter DNA?


Since proteins don't last very long, they are poor targets for
genetic engineering.


Changes in DNA can be carried over generations, making
it a good target for lasting changes.


There are two basic types of modifications.


A) addition

-

to add a function to a cell all you have to do
is introduce a new gene that codes for the given function.


B) deletion

-

deletion of function can be performed by
either "knocking out" a gene or introducing an "antisense"
gene to interfere with the cell's ability to express a given
gene.


Tools of Genetic Engineering


Restriction enzymes
-

used to cut DNA at
specific points during production of
rDNA.


DNA ligase

enzyme seals DNA fragments
into the gap created by the restriction
enzyme.


Transfer
of new genetic material back
into the cell
.



T
ransferring Foreign Genetic Material
Into Plant Cells.



Recombinant DNA

uses bacterial plasmids and viruses
to transport the new genes into the host cells.


Microinjection

involves injecting the genetic material into
the recipient cells using a tiny glass needle. The injected
genes then incorporate themselves among the host
genes.


Bioballistics

use tiny slivers of metal that are coated with
the genetic material. The slivers are "shot" into the host
cells using a "gene gun". Once inside the cells, the new
genes go to the nucleus and become incorporated with
the host genes.



Cloning Steps



Isolate and fragment of source DNA.


Join source DNA to cloning vector.


Incorporate DNA into host.


Detect and purify desired clone.


Replicate clone to high numbers.


Restriction and Modification
Enzymes



Enzymes that cut DNA in specific places


Function: inactivate foreign DNA which
can derange metabolism


Breaks only palindrom sequences, i.E.
Those exhibiting two
-
fold symmetry
mechanism



Important in DNA research, i.E.
Sequencing, hybridization companies
purify and market restriction enzymes

Organism

Enzyme
designation

Recognition
sequence

Escherichia coli

EcoRI

G A
A
TTC

Escherichia coli

EcoRII


C
C
AGG

Haemophilus
hemolyticus

HhaI

G
C
G
¯

C

Bacillus subtilus

BsuRI

G
C
C

Brevibacterium
albidum

BaII

TGG
C
CA

Thermus aquaticus

Taql

T


CG
A

Examples



Cloned Cows Produce Human
Antibodies


Human Artificial Chromosomes

(HACs) were introduced into
cow cells that were then fused with bovine eggs from which
the chromosomes had been removed.


The embryos were then implanted in cows and allowed to
develop into live calves.


Even though the human genes were floating in their own
chromosome, the researchers found the HAC was faithfully
replicated by 80 per cent or more of the cow cells.


When they examined immune cells from the animals, they
found some had correctly assembled human antibody genes.


Genetic Engineering Plants


Crops are engineered for several reasons.


Popular and successful engineering has
allowed for plants to possess tolerance to:
-
.


-

Herbicides, cheap pesticide used to kill
weeds not harm crops.


-

Insects,


-

Viruses,


-

Diseases,


Flavr
-
savr tomatoes no need to ripen
artificially contain gene that inc shelf life.


Misuse of Biotechnology


Companies


terminator genes


Biological warfare

Human Genome Project


B
egun in 1990, project
goals

were to:
-
.


-

Identify

all the approximately 30,000 genes in human
DNA,


Determine

the sequences of the 3 billion chemical base
pairs that make up human DNA,


Store

this information in databases,


Improve

tools for data analysis,


Transfer

related technologies to the private sector, and.


Address

the ethical, legal, and social issues (ELSI) that
may arise from the project.

Human Genome Project


A working draft of the entire human genome sequence was
announced in
June 2000
,


Analyses published in
February 2001
.


An important feature of this project is the federal
government's long
-
standing dedication to the
transfer of
technology to the private sector
. By licensing technologies
to private companies and awarding grants for innovative
research, the project is catalyzing the multibillion
-
dollar
U.S. Biotechnology industry and fostering the
development of new
medical applications
.



Diagnosing and Predicting Disease and
Disease Susceptibility



All diseases have a genetic component, whether inherited
or resulting from the body's response to environmental
stresses like viruses or toxins.


The ultimate goal is to use this information to develop new
ways to treat, cure, or even prevent the thousands of
diseases that afflict humankind.


In the meantime, biotechnology companies are racing
ahead with commercialization by designing diagnostic
tests to detect errant genes in people suspected of having
particular diseases or at risk for developing them.


The potential for using genes themselves to treat disease
--
known as gene therapy
--
is the most exciting application of
DNA science.



Genes
-

through the proteins they encode
-

determine how efficiently we process foods, how
effectively we detoxify poisons, and how
vigorously we respond to infections.


More than 4,000 diseases are thought to stem from
mutated genes inherited from one's mother and/or
father.


Common disorders such as heart disease and most
cancers arise from a complex interplay among
multiple genes and between genes and factors in
the environment

Mutations


A
hereditary mutation

is a mistake that is present in the DNA of
virtually all body cells. Hereditary mutations are also called
germline mutations

because the gene change exists in the
reproductive cells (
germ cells
) and can be passed from
generation to generation, from parent to newborn. Moreover, the
mutation is copied every time body cells divide.


Acquired mutations
, also known as
somatic mutations
, are
changes in DNA that develop throughout a person's life. In
contrast to hereditary mutations, somatic mutations arise in the
DNA of individual cells; The genetic errors are passed only to
direct descendants of those cells. Mutations are often the result
of errors that crop up during cell division, when the cell is
making a copy of itself and dividing into two. Acquired
mutations can also be the byproducts of environmental stresses
such as radiation or toxins.


Mutations


Mutations occur all the time in every cell in the
body.


Each cell, however, has the remarkable ability to
recognize mistakes and fix them before it passes
them along to its descendants.


But a cell's DNA repair mechanisms can fail, or be
overwhelmed, or become less efficient with age.
Over time, mistakes can accumulate.


Genetic Testing


Genetic tests can be used to look for possible predisposition to
disease as well as to confirm a suspected
mutation
.


Newborn screening

-

most widespread type of genetic testing
some tests look for abnormal arrangements of the chemical bases
in the gene itself, while other tests detect
inborn errors of
metabolism

(for example,
phenylketonuria
) by verifying the
absence of a protein that the cell needs to function normally.


Carrier testing

can be used to help couples to learn if they carry
-

and thus risk passing to their children
-

a recessive allele for
inherited disorders such as cystic fibrosis, sickle
-
cell anemia, or
Tay
-
Sachs disease

(a lethal disorder of lipid metabolism).


Prenatal diagnosis

-

also are widely available for the of
conditions such as down syndrome, beta
-
Thalassaemia.


Genetic Tests


Doctors make use of genetic tests to identify telltale DNA
changes in cancer or precancer cells.


Such tests can be helpful in several areas: early detection
(
familial adenomatous polyposis

genes prompt close
surveillance for colon cancer); Diagnosis (different types
of
leukemia

can be distinguished); Prognosis (the product
of a mutated
p53 tumor
-
suppressor gene

flags cancers that
are likely to grow aggressively); And treatment (antibodies
block a gene product that promotes the growth of breast
cancer).


Much of the current excitement in gene testing, however,
centers on
predictive gene testing
: tests that identify people
who are at risk of getting a disease, before any symptoms
appear. Tests are already available in research programs
for some two dozen such diseases, and as more disease
genes are discovered, more gene tests can be expected.


Gene Therapy


A novel approach to treat, cure, or ultimately prevent
disease by changing the expression of a person's genes.


Current gene therapy is primarily experimental, with most
human clinical trials only in the research stages.


Gene therapy can be targeted to somatic (body) or germ
(egg and sperm) cells.


Many people falsely assume that germline gene therapy
already is being done with regularity.


News reports of parents selecting a genetically tested egg
for implantation or choosing the sex of their unborn child
may lead the public to think that gene therapy is occuring.
Actually, in these cases, genetic information is being used
for selection. No cells are altered or changed.




First Actual Case of Gene Therapy





Deficiency of the protein adenosine deaminase "ADA“,
involved in maturation of T and B cells.


Causes collapse of the immune system.


Remove stem cells or WBC’s by taking blood samples.


Culture cells & genetically engineer with recombinant
DNA containing good ADA gene using a viral vector.


Reintroduce cultured cells into host (transfusion.


New stem cells and /or WBC’s make ADA and immune
system recovers.


This procedure to recover function of ADA only worked
because blood cells are easy to remove and to
reintroduce.



Gene Therapy Trails



Children with severe combined immunodeficiency
syndrome (SCD) underwent
ex vivo

gene therapy.


Gene therapy trials include treatment of
hypercholesterolemia where liver cells lack a receptor
for removing cholesterol from blood.


Cystic fibrosis patients lack a gene for trans
-
membrane chloride ion carriers; patients die from
respiratory tract infections. Liposomes, microscopic
vesicles that form when lipoproteins are in solution,
are coated with healthy cystic fibrosis genes and
sprayed into a patient's nostrils. This method is
in
vivo

method.


PCR


Usually only tiny amounts of DNA can be isolated from
individual living organisms, too little for engineering
purposes.


DNA can be amplified by "cloning“, i.e. adding
recombinant DNA to bacteria and grow up many kg of
bacteria.


A more recent technique is the polymerase chain reaction
or PCR.


Millions of copies of a DNA fragment can be made in a
few hours entirely
in vitro
.


Uses DNA polymerase, the enzyme normally used by
bacterial cells to make copies of DNA in the cell cycle.


Can amplify tiny amounts of DNA from fossils, embryonic
cells, tissue or semen samples from crime victims.


"Jurassic park."



Genes exist in different forms called alleles.


When cellular DNA is cut with restriction enzymes they
form restriction fragments of different sizes depending on
the alleles present.


Different sized fragments when separated on a gel show
"restriction fragment length polymorphisms" (RFLPS).


Since each individual has different alleles for their genes,
each individual will show a characteristic "DNA
fingerprint" .


Hasn’t always been acceptable as a form of proof of guilt
in criminal investigations but is very useful in eliminating
suspects.


Has been used to identify rapists and murderers and in
some cases to release innocent persons from prison
.



DNA Fingerprinting

Mitochondrial DNA Typing


When nuclear DNA in small amounts.


Hundreds of copies of mtDNA for every copy of nuclear DNA.


Maternally inherited.


Used to identify remains of the Russian Royal family.


Nine skeletons had been found in a shallow grave in
Ekaterinburg, Russia, in July 1991.


Mitochondrial DNA was obtained and sequenced from the
remains by a team from Moscow, Cambridge and the UK FSS.


The skeleton thought to be that of the Tsarina did indeed have
the same sequence as the three thought to be her children.


Furthermore, the sequence also matched that from the Duke of
Edinburgh, who is related through the female line to the Tsarina
and who would therefore be expected to have the same
mitochondrial DNA



Ethical Questions Concerning
Genetic Engineering on Humans





When and on whom should genetic testing be done?


Who should have access to results of genetic testing?


Should people be denied employment or insurance because
they have genetic profiles deemed "defective"?


With spiraling medical costs, should carriers of genetic
disorders be denied the right to have children? Forced to
pay high insurance premiums?


Do humans have the right to produce and patent new
transgenic species?


What about medical researchers patenting genetic material
from cells derived from your body with or without your
consent?