chromosome manipulations

A N I M A L B I O T E C H N O L O G Y

Chromosome Manipulations

Benefits of Genetically Engineered Animals

Used to develop new medical treatments

Improve our food supply

Enhance our understanding of biology of all animals, including humans

Animal Models

Animal systems are a model for the human system

Polio vaccine was developed using animals as test systems

Cataract surgical procedures were developed with animals

Dialysis was tested first in animals before being applied to human conditions

Regulation of animal research

Animal Welfare Act

Sets specific regulations regarding, housing, feeding, cleanliness and medical care of animals

Researchers must first develop a plan describing

Appropriateness of species to be used

Minimum number of animals needed for test

Oversight committee reviews and approves plan

Government agencies monitor welfare of the test animals

Phase Testing

Testing a new product for safety in humans involves vigorously following scientific methodology developed for animal systems

Involves collecting data from a statistically significant number of trials (experiments) in lab cell tissue cultures, in live animals and in human subjects.

3-stages of testing

Tissue culture

Animal

model

Human trials

if

successful

if

successful

Testing

If test results using cell cultures indicates toxicity of product, then product will never be tested on live animals.

Testing on live animals requires evaluation of more than one species, since different species may respond differently.

Phase Testing

Animal models can provide the following information on a new product

Absorption of chemical by body

Body metabolism of chemical

Time require for chemical or product to be excreted

If significant problems are encountered with product in live animals, then product is never tested in humans.

Side-effects of new drugs discovered in animal models

Example Propecia

Used to encourage hair growth

Animal studies indicated that serious birth defects occurred in male offspring when pregnant animals were given large doses of drug

As a result of animal tests, warnings were put on containers of Propecia to avoid birth defects in humans using drug.

How do you select appropriate animal as a model for the human system?

Look for genetic homology between animal and human systems.

In addition, identify animal that

Has short time between generations

Can produce lots of offspring in each generation

Can be easily maintained and manipulated in the laboratory

Matching animal systems as models for the human system

Lung and cardiovascular

Immune system

HIV and AIDS research

Dog

Mice

Monkey and chimpanzee

System Best animal model

for human

A model organism is a non-human species that isextensively studied to understand particularbiological phenomena

Enviro Pig TM

Transgenic pigs express phytase in their salivary glands

Phytic acid in the pig meal is degraded releasing phosphorus

The phosphorus is absorbed by the pig.

Normally the phytic acid/phosphorus complex passes through the pig and is excreted as waste

Pig waste is a major pollutant & can cause eutrophication of lakes & streams.

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transgenic fish

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Laboratory ratA laboratory rat is a rat of the species Rattus norvegicus (brown rat) which is bred and kept

for scientific research. Laboratory rats have served as an important animal model for

research in psychology,medicine, and other fields.

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Zebrafish

Zebrafish

Lots of genetic similarity to humans

Egg lends itself to genetic transfer no need to implant an egg inside a donor mother for gestation.

Embryos are transparent, making it possible to study cell division under microscope from first hour of creation. transplant gene into embryo

Because the genetics of zebrafish and humans are similar, they are ideal animal systems for determining whether a new drug induces genetic mutations

Easy to follow

drug effect on

embryo

development

under

microscope, sinc

e egg can

mature outside

female.

Homology Testing

Oxford Grid

Dots represent similar genes

Boxes with more than one dot

represent conserved sequences

human

Exchanging genes between individuals

Somatic cell of human

Chromosome 5

Targeted gene disruption or insertion

Homologous

Recombination

(rare event)

Cloned in tissue culture

Select for recombinant

before somatic cells

stop dividing

Reconstructed

embryo

Look for effect

of gene disruption

or insertion on

organ development

Homologous Recombination

flawed gene

good gene

Person 1

chromosome

Person 2

chromosome

Exchange section of DNA on one chromosome with

a section of DNA containing good gene on another

chromosome.

gccatt ccgtc

cggtaa ggcag

gccatt ccgtc

cggtaa ggcag

Offspring now has a copy of good gene from Person 2 in

allele donated from Person 1

Mix chromosomes

and promote DNA

replication by

mitosis.

Reconstructed embryo

Nucleus from

somatic cell

Functional

tissue or organ

Egg divides to produce

differentiated cells

An new clone, a genetic

copy of the donor, forms

when the egg starts to

divide

Step 2: insert nucleus from transformed cellGenetically

modified

somatic cells

Embryo Reconstruction

EMBRYO RECONSTRUCTION BY

TRANSPLANTATION OF THE DONOR

INNER CELL MASS TO THE RECIPIENT

BOVINE BLASTOCYST

Embryo reconstruction for chimera production

has been used in experiments oriented towards

animal science such as the production of

interspecies pregnancies in domestic animals . So

far, chimeras have been obtained by the

aggregation of the blastomeric or by inner cell

mass transplantation . The aggregation of cell

from embryos results in embryos with a more

randomly distribution contribution of cells from

each donor to the trophoblast and ICM. in their

production of interspecific sheep-goat chimeras

produced one kid by the injection of a goat ICM

into a sheep blastocyst and one lamb from the

reciprocal injection

Nuclear Transfer

Remove the nucleus from an egg

Suction

to hold eggPerforate egg with

needle and withdraw

intact nucleus

egg

Step 1:

C R E A T I N G D O L L Y : A B R E A K T H R O U G H I N C L O N I N G

Cloning

Embryo twinning (conventional approach)

splitting embryos in half to produce artificially created twins

commonly practiced in cattle industry today

limitation is that organisms being copied is unknown

you may or may not end up with an animal that has the desired characteristics and you have to wait until the animals is full-grown to find out.

Dolly was created from an adult cell-not an embryo

Dolly was an exact copy of an adult with known characteristics.

How is this done?

Cells collected from donor animal and put in a

culture medium that keeps them alive but prevents

their replication and stops gene

expression.

Egg of an animal has it’s nucleus (DNA) removed

(enucleation)

Nucleus of cultured somatic cells from donor

animal are then inserted into a recipient animal’s

egg next to its cytoplasm.

Apply low-level electric charge and fuses with egg

cytoplasm to produce a 1-cell cloned embryo.

New cell containing egg behaves as if it were an

embryonic cell rather than an adult cell. Cell

division occurs just as it would in an ordinary

fertilized egg.

Transfer embryo to surrogate mother for gestation.

Newborn will be genetically identical to donor

Successfully cloned species

Sheep

goat

pig

cow

endangered cow (gaur)

house cat

gaur

Limits of cloning

Viable cell is required

Success rate is still low

Dolly was successful only after 277 failed attempts

only 29 implanted embryos lived longer than 6 days

Many clones are born with defects

kidney problems

diabetes

crippling disabilities

old before their time-telomere length

Dolly was diagnosed with arthritis -premature aging?

Cloning as a means of producing replacement body parts?

Idea is to reduce chance of cloned tissue from being rejected by original “parent”.

It would take years for clone to produce the organs to be used for transplant

Benefits of Cloning

Reduce variability of responses of a population being used to test new drugs, etc.

avoids confounding factor of different genetic predispositions

Preservation of endangered species

cloning pandas using common black bear as surrogate host.

Reduce time to produce new breeds of farm animals

from 6-9 years 3 years

Early experiments on transgenic animals

A new gene was added to a cell grown in a tissue culture and the effects on that one cell were observed.

With the introduction of cloning, a gene could be added to many cells, and all the cells could be screened to see which one(s) contained the gene.

Each cell that contained the gene could then be used to grow a complete animal using cloning technology

Transgenic techniques

Retrovirus-mediated transgenesis

infect mouse embryo with retroviruses before the embryos are implanted into an animal for gestation.

Retrovirus acts as a vector for the new DNA

size of new DNA is limited

viruses genetic material can interfere with embryo development

not very efficient

embryo

cell

nucleus

retrovirus

Pronuclear injection

Introduction of foreign DNA at earliest possible stage of development of the zygote (fertilized egg)

Just before the egg and sperm cells join, DNA is injected into the nucleus of either cell.

Since the DNA is injected with a syringe, no vector is required and no vector genetic material is introduced that could complicate outcome

Embryonic stem cell method

Embryonic stem cells are collected from inner cell mass of blastocytes

Cells are mixed with foreign DNA

some cells take up the foreign DNA and incorporate it into cell’s own DNA in the nucleus and are “transformed”

Transformed cells are injected into the inner cell mass of the host blastocyte for differentiation and development

blastocyte

Foreign DNA

Transformed

cell

blastocyte

Transgenics to make milk healthier for humans

Lactoferrin-protein that binds iron needed by human babies for development

introduce gene for this protein into cells of cow that are responsible for milk production

Human immune genes introduced into cows as a factory for human antibody production.

Transgenics as a means of deleting genes and their functions

Deleting a gene is a way of determining what its function is in the cell

Active gene is replaced with a gene that has no functional information

When the gene is “knocked out” by the useless DNA, the trait controlled by the active gene is eliminated from the animal

.

Knockout Mice

Knockout mice begin as embryonic stem cells with specifically modified

DNA that has been prepared by recombinant techniques. The

modification results in a nonsense mutation in the normal gene of the

animal.

Homologous recombination within target gene

normal gene

Useless DNA

Chromosome

with normal

gene

Plasmid with

useless DNA

insert section of DNA of gene on vector into

a section of DNA containing good gene on

chromosome of stem cells.

gccatt ccgtc

cggtaa ggcag

gccatt ccgtc

cggtaa ggcag

Chromosome is modified with a useless form of the gene. Look for a trait that has

changed

Recombination

between vector

and chromosome

Random insertion of useless gene at a location other than the target gene

normal gene

Useless DNA

Chromosome

with normal

gene

Vector with

useless DNA

Insert section of DNA of useless gene on vector into

a section of chromosome that does not disrupt target gene.

gccatt ccgtc

cggtaa ggcag

gccatt ccgtc

cggtaa ggcag

Chromosome is modified with a useless form of the gene at some other site than target

gene

Recombination

between vector

and chromosome

BlastocyteTransformed stem cell

Knockout

mouse with

nonfunctional

gene in all its

differentiated

somatic cells

chimera

Not all cells had the

trait changed

Need to crossbreed

for 2 generations to

get all cells to lose

trait.

Producing human antibodies in animals

Antibodies are proteins whose structure gives it the ability to bind very specifically to other proteins

Antibody (Ab)

Antigen

(Ag)

Region of antigen

protein that is specifically

recognized and bound by

antibody

Antibodies could be designed that target and inactivate cancer cells in our bodies.

Myelomas: antibody-secreting tumors

Monoclonal Abs (mAb) are produced from myeloma cells that produce an Ab that reacts with only one region of an antigenic protein

Making cells that produce

monoclonal antibodies

Once a cell line is identified

that produces an antibody

against a specific antigen, it

can be replicated and the cells

frozen until needed to make

the specific antibody

The specific antibody is

released into the culture

medium and recovered

Review

Approaches to change genomes of animals

Nuclear transfer of genetically modified somatic cell into an egg. Rapid growth of organs for transplant into donor animal.

Nuclear transfer of somatic cell into egg implant into surrogate to produce viable organism (Dolly)

Retrovirus mediated genetic modification in animal genome.

Nuclear transfer of embryonic stem cell into egg.

Implant into surrogate to produce viable

organism