Biotechnology

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9 Δεκ 2012 (πριν από 5 χρόνια και 27 μέρες)

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Describe the science of Biotechnology and its
product domains



List the steps to producing a GMO through use
of
rDNA



Outline the steps of producing and delivering a
product made through recombinant DNA
technology



Identify the important labs and agencies in the
US, and what they’re mainly responsible for



Apply the strategy for values clarification to
bioethical issues



Identify products made through biotechnology



Identify products made through biotechnology


Insulin


Biotechnology


DNA


rDNA

technology


PCR


Cloning


Fermentation


Diabetes


Antibiotics


Restriction enzyme


DNA
ligase


rDNA


Proteases


Antibodies


Pharmaceutical


R&D


Pure Science


NIH


CDC


DNA Fingerprinting


Microbial agents


Virus


Applied science


Moral


GMO


E. coli


Plasmid


t
-
PA


Efficacy


Clinical trials


FDA


Cystic fibrosis


Biochemistry


Molecular biology


Genetics


ethics


Therapeutic


EPA


USDA


Data


Hypothesis


Variable


Control


Positive control


Negative control


Concentration


Journals


Genome


bioethics


We have been manipulating entire organisms
for thousands of years through selective
breeding to produce plant and animal products
that are


Bigger


Healthier


More productive


Have novel phenotypes



Scientists recently learned to manipulate not only
whole organisms, but also the molecules, cells, tissues,
and organs of which they are built


Insulin is a protein produced in healthy
individuals that facilitates the uptake of sugar
from the blood stream into specific tissues


In some diabetic patients, the ability to produce
insulin is impaired


In the past diabetic patients had to take
injections of Bovine (cow) insulin to regulate
their blood sugar


Problem: getting insulin from cows is not very
efficient, it’s time consuming and expensive,
and can lead to improper immune stimulation
of the recipients


Solution: Biotechnology


We have learned to manufacture large amounts of
specific molecules, such as
human

insulin in bacterial
cells


Cost effective


Convenient


Fast

PAST

PRESENT


Selective breeding


Fermentation: a process
by which in an oxygen
deprived environment a
cell converts sugar into
lactic acid or ethyl
alcohol (ethanol)


DNA technology


Recombinant DNA
(
rDNA
) technology


PCR


Cloning


Proteomics



Since biotechnology is constantly changing
there is no one definitive definition


In 2003 in an international treaty on
biotechnology, modern biotechnology was
defined as :


“the application of techniques that overcome natural
physiological reproductive or recombination barriers
and that are not techniques used in traditional
breeding and selection “ including


In vitro nucleic acid techniques


The fusion of cells beyond taxonomic family


One major discovery that propelled the
biotechnology science and industry was the
discovery of restriction enzymes


Restriction enzymes: enzymes that cut DNA at
specific recognition nucleotide sequences
known as
restriction sites



They were discovered in bacteria & are
thought to have evolved to provide a defense
mechanism against invading viruses



Restriction enzymes allow us to cut and recombine
DNA molecules, often from different species



“cutting”


Reminder


DNA (deoxyribonucleic acid) is a double
stranded helical molecule that that stores genetic
information for the production of all of an organisms
proteins


DNA
Ligase
: an enzyme that binds the
disconnected strands of DNA together



“pasting”


If you put DNA into an organisms cell, that cell
will produce the protein that DNA codes for!




Advances in
rDNA

technology allow
biotechnologists to manufacture a wide variety
of products either previously unavailable or
that could only otherwise be made in small
quantities


Examples:


Insulin
: for treatment of diabetes


Proteases
: function in breaking down other proteins


Antibodies
: proteins normally developed by the
immune system for recognizing specific molecules
called antigens on infectious agents and so limiting
their infectious ability


PCR: Polymerase Chain Reaction is a technique
that involves copying short pieces of DNA and
using them as templates to make millions of
copies in a very short amount of time



Cloning: method of asexual reproduction that
produces identical organisms


Done mainly in bacteria and some fungi


Only happens in animals if manipulated by scientists
to do so


Cao
Yilin
, a plastic surgery expert in Shanghai,
successfully reproduces a human ear on the
body of a little white mouse on Friday, April 4,
1997 in Shanghai.

Dolly, right, the first cloned sheep
produced through nuclear transfer
from differentiated adult sheep
cells, and Polly, the world's first
transgenic lamb, are in their pen at
the
Roslin

Institute in Edinburgh,
Scotland, in early December, 1997.


Biotechnology is practiced in a variety of
settings , and in general the setting determine
the major emphasis


Private companies: focus is making money so they
tend to emphasize the development and
manufacture of products that will yield high profit
margins


The do have a Research & Development department,
but they will only fund research that has potential for
financial pay off


Universities & Government agencies focus on
research


More involved in Pure science




The main difference between
companies and university or
government labs is that companies
must provide a product or service
that results in earnings, a nonprofit
research facility does not



Pure
-
science: Scientific research whose main
purpose is to enrich the scientific knowledge
base



This is the type of science nonprofit labs focus on


Applied Science: the practice of utilizing
scientific knowledge for practical purposes,
including the manufacture of a product


This is the type of research
forprofit

labs focus on



Pharmaceutical


Agricultural


Industrial


Research or production instruments, reagents,
or data


Some companies sell their services rather than
product


Example:
23 and Me


See figure 1.13


Research & Development : refers to the early
stages of product development that includes
discovery of the structure and function of a
potential product and the initial small scale
production


Quality Control is a measure of precision or
how well the measurement system reproduces
the same result over time and under varying
operating conditions


National Institute of Health (NIH): the federal
agency that funds and conducts biomedical
research



Centers for Disease Control & Prevention
(CDC): National research center for developing
and applying disease prevention control,
environmental health, and health promotion
and
educationactivities

to promote public
health


By the 1970’s scientists had developed new
methods including the use of restriction
enzymes and DNA
ligase

to create new
combinations of genetic information, or
rDNA


rDNA

can be inserted into cells giving these
cells a new genotype phenotype


Organisms containing these modified cells are
called Genetically Modified Organisms
(GMOs)


GMO’s contain DNA from another organism
and will produce the new proteins encoded in
the acquired DNA


The 1
st

GMO’s to produce a human protein
were
Escherichia coli
(
E. coli
) bacteria


E. coli
bacteria are the work horse of molecular
biology, no other bacteria is used as frequently
as this one


Were given pieces of human DNA (genes)
containing instructions to produce a human
growth hormone called
somatostatin


The
somatostatin

gene was carried into the E.
coli cells on tiny pieces of bacterial DNA called
Plasmids


These recombinant DNA plasmids contained both
bacterial and human DNA


The E. coli cells read the human DNA and produced
the human protein
somatostatin
, which could then
be harvested and purified for use


One of the 1
st

genetically engineered products
to be sold was t
-
PA


t
-
PA is a blood clot dissolving enzyme that can
be used immediately after a heart attack to
clear blocked blood vessels


The body produces only small amounts of t
-
PA


To produce enough t
-
PA for therapeutic use
scientists genetically engineered mammalian cells
using Chinese Hamster Ovary (CHO) cells


Ovary cells are grown in culture and
engineered with the gene that provides the
instructions to make the t
-
PA enzyme


By subjecting the cells to the right conditions,
the CHO cells accept and incorporate the
foreign DNA and begin the process of
transcription and translation


Large amounts of t
-
PA can then be extracted
and purified


See figure 1.18


Applications of
rDNA

and genetic engineering
have resulted in the creation of hundreds of
Biotech companies , specializing in all kinds of
GMOs and their protein products including


Proteins used in pregnancy tests


Enzymes that increase the amount of juice that can
be extracted from an apple


Molecules used in vaccines


In 2004 there were already over 370 drug products
created from
rDNA

techniques undergoing clinical
trials


Strawberry plants that can grow in freezing weather



Biotechnological advances will enable patients
to submit tissue samples and receive a
treatment regimen designed specifically for
them. This is
calle

Personalized medicine, or
pharmacogenomics


pharmacogenomics
: pharmaceuticals based on
genomics


Present research based on broad characteristics such
as ethnicity, gender, and family history has already
been performed and treatments have been
modofied

accordingly


The human genome project was an
international effort to sequence and map all the
DNA on the 23 pairs of human chromosomes


We know the sequence of the human genome,
but we still do not know


all of the sequences that code for proteins


For the ones we do know about, we don’t completely
understand how, when, and where they are turned
on or off


This is called
differential gene expression


DNA intro


As sequencing human genomes becomes more
cost effective, there will come a time when
drug regimen is determined by the genotype of
the patient or the tumor being targeted


One of the 1
st

drugs to accomplish this is the
drug
Herceptin

which will only target breast
cancer cells that produce too much of the
protein HER2


The decision to administer
Herceptin

as part of
the chemotherapy regimen is based on a single
gene


Some drugs, like chemotherapeutic agents,
have serious side effects because they treat the
whole organism and not just the diseased
tissue


Advances in biotechnology have enabled the
development of therapies that can be delivered
directly to diseased tissue and greatly reduce
side effects


Rexin
-
G: a drug that targets pancreatic cancer is
delivered by a genetically engineered viral particle
that is designed to seek out and kill metastasized
cancer cells is in clinical trials now


Research & Development


No matter what the product, the goal is to
make it and market it as quickly as possible


The R&D phase for a new pharmaceutical often
takes from 10
-
15 years for a company to move
a product through the “pipeline”


A drug must demonstrate “proof of concept”
data in the research phase before being allowed
to move to the development phase, including


Is it feasible to manufacture in amounts sufficient to
treat people


What needs to be done to ensure safety


What characteristics indicate it will be effective


And unfortunately, what is the profit margin


This is accomplished with a Product development
Plan


Does the product meet the following criteria


Does the product meet a critical need?


Is there a market large and wealthy enough to produce
enough sales


Does preliminary data support that the product will
work?


Can patent protection be secured


Can the company make a profit?


Each product in a pipeline will be reviewed
regularly and dropped from production if the
answers to these questions are unsatisfactory


See figure 1.21


Projects then enter
clinical development


Testing is done small scale before large scale
production is pursued


All pharmaceuticals have to undergo at least 3
rounds of strict clinical trials under the guidance of
the FDA before being marketed


Example:
Pulmozyme

is manufactured by
Genentech Inc and is an enzyme used to treat
patients with Cystic Fibrosis


People with CF experience a build up of mucus in the
lungs and intestines and is often fatal by the age 30


Pulmozyme

reduces the amount of mucus produced


In 2003
Pulmozyme

had sales of
$167million


All biotechnology products have regulations
governing their production in the product
pipeline


Environmental Protection Agency (EPA)


The federal agency that enforces environmental laws
including the production and use of microorganisms,
herbicides, pesticides, and GMOs


United States Department of Agriculture (USDA)


Federal agency that regulates the use and production of
plants, plant products, plant pests, veterinary supplies
and medications, and genetically modified plants and
animals


Being able to distinguish between right and
wrong and to make decisions based on that
knowledge is considered having good morals


Problem: not all people have the same list of
right and wrong, and so we often have
different morals


Example: Vegans don’t eat or use any animal
products, and many consider it immoral to do
so


Fact: most people eat and use animal products
on a daily basis without any qualms

Also Fact: those same
people that don’t have
any problem eating meat
and using animal
products would be
horrified to think of
going home and cooking
their pet dog Sparky or
their new kitten Mittens
for dinner


The study of moral standards and how they
effect conduct is called ethics



Bioethics is a term that has been coined to
describe the study of decision making as it
applies to moral decisions that need to be made
because of advances in biology, medicine, and
technology


Many of the new biotechnologies are
controversial because they force people to
think about what they believe is right or wrong


Examples:


Harvesting and manipulating embryonic stem cells


It kills the human embryo


Genetically modifying food


Are they really safe to eat


Prenatal genetic testing/ engineering


Playing God



New technologies generate ethical questions


The positions one takes on ethical issues are
based on how you feel about the knowledge
and understanding you have of a subject


There is often not a clear right or wrong answer


Most of the time you have to make a choice
between the lesser of 2 evils


So how do we decide what’s “right”

But sometimes people are!


Identify and understand the problem or issue.


List all the possible solutions to the issue


Identify the pros and cons of each of the
solutions


Consider legal, financial, medical, personal, social,
and environmental aspects


Based on the pros and cons for each solution,
rank all the solutions from best to worst


Decide if the problem is important enough to
take a position. If it is, decide your position and
be prepared to describe and defend it


There is no 1 correct scientific method


The scientific method is really just a series of
common sense steps and techniques that
scientists use to solve a problem or answer a
question


There are several practices used by most
scientists when conducting experimental
research


State a testable scientific question or problem
based on some information or observation


Develop a testable hypothesis


Plan a
valid

experiment


Multiple replicates


Control groups


Conduct the outlined experiment and collect
and organize data


Formulate conclusions based on experimental
data and error analysis

POSITIVE CONTROLS

NEGATIVE CONTROLS


A group that will give a
predictable positive
result


A group that will give a
predictable negative
result


A good approach to writing an experimental
conclusion is to use the REE/PE/PA method


REE:
Results

with
Evidence

and
Explanation


PE:
Possible Errors


Identify the sources of
expreimental

design errors that
would or could lead to false or misleading data, and
explain possible implications from such errors


PA:
Practical Applications:


discuss the meaning or value of experimental results in
the short and long term.


Identify how the findings could be valuable clinically, to
the company, or to the community


Identify the direction the experiment will go take from
here



Biotechnology: The study and manipulation of
living things or their component molecules,
cells, tissues, or organs


Product Domains


Pharmaceutical


Agricultural


Industrial


Research or production instruments, reagents, or
data



Identify a gene of interest


Treat gene of interest and plasmid vectors with
a restriction enzyme


Use DNA
ligase

to glue gene of interest to
plasmid


Insert plasmid into host organism (plant,
animal, or bacteria) through various
mechanisms


The
rDNA

will be used by the host organism to
produce the new protein of interest


Come up with an Idea


Research and Development


Proof of Concept through a Product Development
Plan


Will it work


Is there a market for it


Can we make it in sufficient quantities


Large Scale Production


Clinical Trials


FDA approval



NIH: the federal agency that funds and conducts
biomedical research


CDC: National research center for developing and
applying disease prevention control, environmental
health, and health promotion and education activities
to promote public health


EPA: The federal agency that enforces environmental
laws including the production and use of
microorganisms, herbicides, pesticides, and GMOs


USDA: Federal agency that regulates the use and
production of plants, plant products, plant pests,
veterinary supplies and medications, and genetically
modified plants and animals




Identify /understand the issue


List all the possible solutions


Identify the pros and cons of each considering



legal


financial,


Medica



personal


Social



environmental aspects


Rank solutions from best to worst


Decide and defend your position



T
-
PA


Human insulin


Strawberry plants that can grow in freezing
weather


Pulmozyme