AdvGentech4.ppt

igocheddarBiotechnology

Dec 14, 2012 (4 years and 10 months ago)

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1

Transgenic Animals and
Plants

-

Genetic Engineering of plant
-
> Transgenic plants


-

Genetic Engineering of animals
-
> Transgenic animals

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Definition of Transgenic

Transgenic
-
> stable introduction of a gene into another organism


-
> For Unicellular organisms (such as bacteria or yeast)


all transformed cells are
-
> transgenic


-
> For multicellular organisms (such as animals, plants,..)


difference between:


-

manipulation of single cells
-
> cell line


(expression in insect cells or mammalian cells)


-

manipulation of a whole plant or animal
-
> transgenic


(can have a transgenic offspring!!!)


-
> more difficult and expensive to create whole modified organism
(transgenic) than just cell line!!!

3

Transgenic versus Cloning


Transgenic
-
> creation of transgenic animal or plant (introduction
of foreign gene into organism)


-
> transgenic organisms produced by introduction of foreign gene into germ line


(
-
> transgenic offspring!!!)


-
> introduction of gene into somatic cells
-
> gene therapy


Cloning
-
> obtaining an organism that is genetically identical to the
original organism


-
> such as Dolly the sheep


-
> asexual propagation of plants (taking cuttings)

4

Transgenic Plants

Why do we need transgenic plants ?



improvement of agricultural value of plant (resistance to herbicides,
resistance to insect attack
-
>
Bacillus thuringiensis

toxin)


living bioreactor
-
> produce specific proteins


studying action of genes during development or other biological
processes (knock
-
out plants, expression down
-
regulated)


5

Transgenic Plants


Advantages:

-
Plant cells are totipotent
-
> whole plant can be regenerated from
a single cell (engineered cells
-
> engineered plants)

-

Plants have many offspring
-
> rare combinations and mutations
can be found

-
Transposons used as vectors



Disadvantages:

-
Large genomes (polypoid
-
> presence of many genomes in one
cell)

-

plants regenerating from single cells are not genetically
homogenous (genetically instable)


6

7

Gene


transfer methods



8

Agrobacterium tumefaciens

mediated transfer

9

Ti Plasmid

10

Integration of T
-
DNA into the plant chromosome


-
> Tumor formation

11

Recombinant Ti plasmid
by recombination

Gene transfer
by
cointegration


12

Microprojectile bombardment



“Shotgun”

13

Viral Vectors

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Transfer into protoplasts

Gene transfer across a
protoplast membrane is
promoted by some chemicals
such as polyethylene glycol

Vector + polyethylene
glycol

15

Electroporation

16

Control elements on vector

Frequently used promoter:
-
> 35S promoter from cauliflower mosaic virus

17

Alterations in plant RNA


downregulation of
specific genes

18

Selection marker free transgenic plant

-
> Transposons

19

Applications for engineering plants


Development of Insect
-
, pathogen
-
, herbicide
-

resistant plants


Flower pigmentation


Modification of nutritional content


Modification of taste and appearance


Bioreactor


Vaccines (Cholera toxin
-
like protein in potatoes)


Plant yield (alteration of lignin content
-
> paper industry)

20

Development of Insect
-
, pathogen
-
,
herbicide
-

resistant plants

Toxin from
Bacillus thuringiensis

21

Development of Insect
-
, pathogen
-
,
herbicide
-

resistant plants

22

Development of Insect
-
, pathogen
-
,
herbicide
-

resistant plants

-

Inhibit the uptake of the herbicide

-

overproduce the herbicide
-
sensitive target protein (Glyphosate)

-

reduce ability of target protein to bind herbicide (cyclohexanediones)

-

plant can degrade herbicide (Bromoxynil, Glufosinate, Cyanamide,..)

Manipulations that make a plant herbicide resistance

23

Development of Insect
-
, pathogen
-
,
herbicide
-

resistant plants

Fungus
-

and Bacterium
-

resistant plants

Engineering of plants
-
> express
antimicrobial peptides

24

Flower pigmentation

CHS
-
> Chalone
synthetase
-
> enzyme in
biosynthetic pathway of a
purple pigment

25

Changed nutrition content

-

Amino acids (to increase lysine content in the future in animal food)

-

Lipids (possible to change degree of unsaturation, chain length)

-

Vitamins (Vitamin E, increase Vitamin A in rice)

26

Modification of taste and appearance

Engineer potatoes
-
> produce more glucose and fructose at
higher temperatures

27

Plants as bioreactor

-
Therapeutic agents

-

Antibodies

-

polymers (PHB)

28

Transgenic Animals

Transgene
-
>
Gene coding for a
growth hormone

29

Transgenic Animals

Why do we need transgenic animals ?



living bioreactor
-
> produce specific proteins in the milk
(
cattle, sheep, goats, pigs
)


studying action of genes during development or other biological
processes (knock
-
out animals, expression down
-
regulated)
-
>
models for studying human diseases
-
>
mice


improvement of agricultural value (
fish, bird
)

30

Gene
-
transfer methods


Microinjection


Retroviral method


Engineered Embryonic Stem Cells (ES) method



Knock


out methods (Cre
-
LoxP system)
-
> studying gene
expression + development

31

The first days of an embryo

Embryonic
stem cells
(ES)

Used for retroviral infection

Fertilized egg

32

Microinjection


into the germ line
-
> transgenic animal

Gene injected into the male pronuclei

33

Efficiency of the transgenesis process after
DNA microinjection

34

Retroviral vectors


into the germ line (8
-
cell embryo infected)

-
> transgenic animal

35

Gene Therapy


gene transfer into somatic cells

Viral gene
transfer into
somatic stem
cells
-
> gene
therapy

36

Engineered Embryonic
Stem Cells (ES)


into the germ line (blastocyst)

-
> transgenic animal

Inner cell mass (ICM)

of blastocysts can
form all cells of the embryo
-
>
Pluripotent

-
> Embryonic stem cells

Engineered ES
-
> can form any kind of cell in an
embryo

37

Gene Transfer
-

what happens on DNA level


Integration into chromosome
-
>
Recombinantion


Recombinantion can be
-
>
homologous


non
-
homologous

-

non
-
homologous event
-
> more frequently

-

homologous event
-
> less frequent but
desired



Knock
-
out mutants

-
> disrupt functional gene by integration of
another gene into target gene

Used for:


-
> study human diseases by creating model organisms


-
> make minus mutant

38

Homologous recombinantion

39

How do check for homologous recombinantion

40

Construction of a disruption construct

41

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43

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Cre
-
LoxP system:

-

Inactivation of a gene (knock
-
out) in a specific cell type

-

Activation of a transgene in specific cell type



Used for:

-

Study biological consequences of tissue
-

specific gene inactivation


-
> establishing models for human diseases


-
> selective removal of kinesin II gene (expressed in retinal receptor cells)


-
> leads to accumulation of opsin and arrestin
-
> cell death


-
> result mimics aspects of a disease (inherited retinis pigmentosa)

-
> large deletions in chromosome
-
> deletion in chr. 22
-
> DiGeorge syndrome


(cardiovascular dysfunction)

45

Inactivation of gene in specific cell type (tissue)

46

Cloning of Dolly



Cloning Animals by

Nuclear Transfer Technology

Critical for success
:

Cell cycle of the somatic cells
(udder cells) on plates was critical


they were kept in specific growth
stage (diploid stage)


Of the
434

fused oocytes created
during the experiment
-
> only Dolly
survived to adulthood


Dolly was real clone (genotype
identical) and could reproduc

Dolly was euthanized 2003
-
>
suffering from progressive lung
disease


Since 1997

-
> cloning of sheep,
cows, mice, cats, other animals done

-
> many of the clones developed
severe
diseases as they matured
.

Until 1997, arrival of
Dolly


not possible to
produce an adult
animal from a nucleus
from an adult
animal
´
s
differentiated cell

47

Cloning of Mammals


Reproductive Cloning

-

Genotype identical

-

Phenotype is not necessarily identical
-
> variation due to
random events and due to environment

48

Why do clones have health problems?

Telomeres are found at the end of
each chromosome.

Shrinking of the telomeric ends of
our chromosomes are a sign of aging
of the cell.

Each cycle of cell division the
telomeres are slightly shortened
until they are too short for further
replication
-
> cell death


Dolly
´
s telomeres (at the age of 3)
have been as short as ones of the
age of 6
-
>
clones age “faster”.

49

Why do clones have health problems?

Differentiated cells have certain methylation pattern.

Cloned animals have abnormal methylation pattern originating from nucleus from
differentiated cells

Some can be “re
-
set” (epigenetic reprogramming) to their undifferentiated
state, some cannot
-
>
faulty gene activation in cloned animal


-
> so few cloned embryos survive

-
> surviving clones have severe health problems

50













Production of
pharmaceutical
proteins
-
> drugs


Problems:


Highly inefficient

Only 20% of the
eggs survive and
only 5% of them
produce product

Transgenic Cattle, Sheep, Goat, Pigs

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Transgenic Cattle, Sheep, Goat, Pigs

-

Protein production: in milk, blood, urin

-

Animals (pigs) with modification of sugars on surface of organs


-
> donor for organ transplants

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Transgenic Cattle, Sheep, Goat, Pigs

53

Transgenic birds and fish

-
> improvement of agricultural value

Transgenic chicken:

-

Resistant to viral, bacterial diseases

-

better feeding efficiency (fast growth, better meat quality, more meat

-

less fat meat, less cholesterol in eggs

-

maybe use of eggs as bioreactors for protein production


Transgenic fish:
-
> to support aquaculture

-

Increase growth rate (growth hormone)

-

resistance to diseases

-

Generation of model systems to monitor health hazard


(screening chemicals if they cause mutations)