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Biotechnology: Transgenesis

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Helen Liu

on 17 March 2016

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Transcript of Biotechnology: Transgenesis

Transgenic Studies
BIOTECHNOLOGY
TRANSGENESIS
is the process of genetic modification of an organism by introducing a gene from another species into it.
New DNA is introduced to the genome and integrated with recombinant DNA technology to gain desired traits and to deactivate undesirable ones.
....................................................................................................................................................................................................................................................................
The foreign DNA is transmitted through the entire germ line in order
for all the cells to be affected by the new genetic information.
ORIGINALLY ATTEMPTED IN THE 1970s, IT RESULTED
IN CHIMERIC MICE DEVELOPED FROM TWO
DIFFERENT EMBRYOS, COMBINED.
BEFORE MENDEL, SELECTIVE BREEDING WAS ALREADY DONE BY FARMERS WITHOUT SPECIFIED KNOWLEDGE.
*
Although traits from both strains of mice were expressed, the "transgene" was not successfully inserted into all the cells of the new mice.
THERE ARE THREE METHODS FOR TRANSGENESIS.
DNA microinjection
Retrovirus-mediated gene transfer
Embryonic stem cell (ES)-mediated gene transfer
DNA MICROINJECTION
IS THE DIRECT INJECTION OF GENES INTO AN ORGANISM.
pronuclei developing
Females are given hormone injections
46-48 hours apart for superovulation
They are mated with fertile males and their eggs are harvested
DNA with the desired gene is injected into the pronucleii of the eggs
The transgene integrates into the haploids' chromosomes before meiosis
The egg is surgically transferred to the oviduct of a pseudopregnant female


A tissue biopsy is taken to determine
if the transgene was successfully
integrated into F1 generation.
MICE ARE POPULAR SUBJECTS DUE TO LOW MAINTENANCE COST, A SHORT GENERATION TIME AND WELL-DEFINED GENETICS.
They are mated with vasectomized males
to support the surgically inserted eggs.
MICROINJECTION WAS THE METHOD
USED FOR TRANSGENESIS.
F I R S T
On average, 15% - 20% of mice that were performed on displayed the transgene.
It results in an over-expression or an under-expression of a gene based on what DNA solution was inserted in the pronucleii.
Due to the procedure, microinjection is flexible and is applicable to a wide variety of species.
H O W E V E R ,
The process is unreliable as one cannot control the
site where the gene inserts itself, or if it is expressed at all.
After F1 generation, meiotic recombination may result in a loss of the transgene altogether, since it is on only a section of a single chromosome.
Mutations occur in 10% of all cases.
RETROVIRUS
MEDIATED
GENE TRANSFER
Transgene is inserted by a plasmid or virus into the host
The retrovirus injects its RNA (genetic material) into the cell
With the virus enzyme reverse transcriptase, it makes a DNA copy of itself
Integrase inserts the virus DNA into the original cell DNA, incorporating it into the cell's genome and modifying the cell for a new gene
There is a higher possibility of transgene expression in this method because of the virus' ability to integrate with cells at a high frequency.
HOWEVER, TRANSGENE TRANSMISSION IS ONLY SUCCESSFUL IF THE RETROVIRUS INFECTS GAMETES.
EMBRYONIC
STEM CELL
(ES)
MEDIATED GENE TRANSFER
A method similar to DNA microinjection done with an in vitro ES cell culture
The transgene is inserted by homologous recombination into an ES cell
Useful as stem cells are undifferentiated and may develop into anything
The ES cells are incorporated into an embryo of an organism
This is the method of choice for gene deactivation, or
the " " method.
knockout
SUCCESS RATES OVERALL ARE
VERY LOW.
THE SUCCESSFUL F1 GENERATION IS
ALMOST ALWAYS CHIMERIC.
&
This is due to the only partial integration of the transgene in the organisms' cells.
The transgene must be successfully inserted into the germ cells for it to carry on to further generations
Chimeras must be inbred for
10-20 generations
before true transgenic animals are developed
The transgenic embryos can be then frozen until implantation.
(The unwanted gene is replaced with an inactive one)
TRANSGENIC ANIMALS
USUALLY HAVE SOME AMPLIFIED CHARACTERISTICS.
This allows them
to be applicable in:
Medical research
Molecular biology
Pharmaceutics
Toxicology
Xenografting
[CERTAIN TRAITS SHOW BETTER]
[GENE EXPRESSION REGULATION]
[TARGETED PROTEIN PRODUCTION]
[TOXICANT DETECTION]
[DEVELOPED SPECIFICALLY]
Animals altered specifically for hormones result in:
An increase in product yield
OR
Modification of physiology/anatomy
OR
Cloning to reproduce bloodlines
ALSO BENEFICIAL,
TRANSGENIC PLANTS
are created to improve on weaknesses
that a species may have.
higher crop/produce yield
improved quality in produce
more resistance to pests and disease
more tolerance to heat, drought and cold
GENE GUN
THE
METHOD
USED ON TISSUE CULTURE OR SEEDLINGS
The desired DNA is created/extracted
Microscopic gold/tungsten particles are
coated with copies of the transgene
DNA is shot into the plant by a gene gun
* the tissue culture cells are put in
a vacuum chamber by the gun and
the metal particles are propelled
at them, using a high pressured
gas released in a sudden burst.
AGROBACTERIUM
THE
METHOD
Agrobacterium
Tumefaciens:
A soil-dwelling bacteria that infects plant cells with its DNA.
Usually invades cells and force metabolite synthesis to occur for own benefit
Its DNA in the plasmid (T-DNA) is directed by virulence genes and enters plants through wounds, which send off chemical signals
T-DNA is spectulated to insert itself into exposed DNA during transcription
Scientists inserted the desired gene into the agrobacterium and removed its original genetic material; the bacteria was used as a vessel.
DESPITE THE ARRAY OF BENEFITS TRANSGENESIS SERVES,
one can argue that by altering organisms at the primary level of life
is as though scientists are
PLAYING THE ROLE OF GOD.
WHERE DO WE DRAW THE LINE?
With plants, the ethics involved are clear and straightforward.
But transgenic animals are a different matter entirely.
How much of this technology should be applied to real industry?

How should their conditions be evaluated and described?
How should animal and human welfare be weighed against each other?
(TO WHAT EXTENT SHOULD WE CREATE ANIMALS SOLELY FOR HUMAN USE)
(AS THEY ARE INEXTRICABLY TIED TOGETHER)
I N A N I M A L S
THE
CCAC
works with
AACs
CANADIAN COUNCIL
ON ANIMAL CARE
ANIMAL CARE
COMMITTEES
to write up guidelines and a code of ethics for transgenic animal creation.
T h e y e v a l u a t e a l l t r a n s g e n i c c r e a t i o n p r o p o s a l s .
The potential benefits of the experiment
The ethical cost and potential animal suffering
A plan for unanticipated suffering and to establish end points
A complete transgenic information sheet

A separate protocol is required to create a new transgenic species.
SUBMITTED PROTOCOLS MUST DETAIL:
DUE TO THE FAST EVOLUTION IN THIS FIELD,
THE GUIDELINES FOR LABORATORY ANIMAL CARE ARE CONSTANTLY BEING REVIEWED.
*
Nevertheless,
transgenesis is progress.
It is allowing for advances in medicine that would not be possible otherwise.
THE FIELD IS GROWING AT A RAPID PACE.
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