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Model for diseases
can produce valuable
pharmaceuticals
information
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can be defined as
modification of the genetic
of an organism
through
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European Laboratory
Animal Associations
as an animal in which there has been a
deliberate modification of its,the
genetic
there has been a deliberate modification of its
genome
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WHY?
Study gene function and regulation
Genome mapping
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•
Test
knock in gene
Knock out gene
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Cancer research
Transposons transferred
from galley fish
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•The genes can be modified to jump
wily-nilly around cells. By putting
them into a batch of
identify which genes or combinations of genes lead to cancer.
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Jumping genes from jelly
fish
•By putting them into a batch of
human or mouse cells, and watching
to see which ones become cancerous
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have been made using
animals
medical advances over the last century
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Biomedical research
a treatments before attempting to diagnose
model of a disease to learn about it and test
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Benefits of transgenic?
Phytase enzyme poultry
animal production
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salmon
•Improved gain efficiency and protein
production
Super-salmon
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salmon
Antifreeze protein in flounder +
Chinook salmon
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Totipotentcy
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Embryonic Stem Cell
Lines
Isolate inner cell mass
(destroys embryo)
Heart muscleKidney
Liver
“Special sauce”
(largely unknown)
Day 5-6
Blastocyst
Inner cells
(forms fetus)
Outer cells
(forms placenta)
Heart
repaired
Culture cells
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SCNT
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Nuclear Transfer
Lamb 6LL3 (‘Dolly’)
Donor: (adult) mammary gland
cell - Finn Dorset ewe
Recipient: enucleated oocyte -
Scottish Blackface ewe
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DNA vaccine
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EX-Vivo Trans genesis
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•1981: First transgenic mouse
–Insertion of hGH into a mouse (Singleton,
1999)
–Production of monoclonal antibodies &
anti-inflammatory agents
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CAST, 2003
has been the most common method
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2. An animal is given hormonal treatment to produce a large number
.
Of embryos, and the embryos are collected from the oviduct.
3. The human gene is inserted into the fertilized egg via
microinjection
4. The transgenic embryo is placed in a surrogate host which gives
Birth to the transgenic animal
5. The offspring is tested for the new gene
1. A human gene responsible for producing a desired
protein Is isolated in a laboratory
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transgenic
•gene transfer is the production of
transgenic dairy farm
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FDA, 2005
Mastitis Disease Resistance
Annie, born March 3, 2000, is a clone of a pure-bred Jersey calf
genes for Staphylococcus aurousbacteria
producing lysostaphin, a protein that kills
Staphylococcus aurousbacteria
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Lysozyme is a protein found in the tears
gene for an antibacterial enzyme found in human breast milk,
Lysozyme inhibits the growth of bacteria by destroying the bacterial cell wall
Treatment infant diarrhea
produce human lysozyme in their milk
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Transgenic goat
milk contains a protein that could be extracted
to make a drug for coronary bypass patients.
now in human clinical trials.
Millie is aHer The protein,, is
called anti-thrombin III
goat.
transgenic
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Steps
•the gene that produces AT III is
sequenced,.
build a synthetic gene and make many copies
and
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then
•The synthetic gene is then attached
to the gene forcasein,which acts as
apromoter gene.This ensemble is
then injected into a.
newly fertilized egg
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then
•. During the first few critical cell
divisions, the gene may become
attached to the goat's DNA.
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then
•If successful, the new gene is now a
transgenic which will become
incorporated into all cells during
subsequent divisions
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then
•The embryo is then transferred to a
surrogate mother
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Off spring
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•Cloning through the ages:
–1952: Cloning via nuclear transfer (frog)
–1989-1990: First mammals cloned
–1995: First cloning via cultured
mammalian cells
–1997: First cloning via adult cells –
DOLLY
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history
•First cloning via transgenic adult cells – POLLY
–1998 – 2000: Cloning of cattle, pigs, mice,
goats and monkeys using adult cells
–2001: First reported cloned human embryo
–2002: First cloned pet
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How many genes are there
E. coli 4.6 Mb 4,288
genes
S. cerevisiae 13.5 Mb 6,034
genes
D. melanogaster 165 Mb 12,000
genes
C. elegans 97 Mb 19,099
genes
H. Sapiens 3,300 Mb 40,000
genes
Phenotypes
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Which of which
•Me can be genetically modified
No N o
No
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Ethics
•Generally scientist must be the
most honest persons
•They know
•This is a blessing of Allah
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In human
•Research on human embryo
•Is illegal
•Some done it behind
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I mean
•Animal model for disease
•Bioreactor for producing
•Therapeutic in milk of
•Goat
•rabbit
•Cow
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Transgenic animals are costly:
20,000-30,000 for one
animal,
and low chances of success. If
successful, one animal can
produce
in its life time 200,000-
300,000 million worth of
drugs.
A herd of 600 transgenic cows
could supply the worldwide
demand
of human serum albumin (used
in the treatment of burns
and traumatic injuries)
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Mention what is in your
list?
•Transgenic
•Transgenic mouse
•Gene expression regulation
•Structure function
•Cause and effect
•In path physiological process
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Mention what are in your
list
•Transgenic mouse
•Transgenic rat
•Transgenic rabbit
•Transgenic goat
•Transgenic chicken
•Transgenic cow
•Transgenic chicken
Transgenic
quail
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Transgenic rabbit
•used as model for cardiovascular
diseases
•Used as a model for AIDS
•Used as model for cancer
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Transgenic rabbit
•The recombinant protein can be
produced
•low cost
•Wide scale
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What rabbit milk
?
•What is can be in rabbit milk
•One of the most attractive protein
•Is alfa glycosidase
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Rabbit
both
bioreactor
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when multiple preferred
analysis be performed on
single sample
blood
chronic studies
long term monitoring
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Transgenic chicken
•to understand normal and abnormal
embryo development
limb deformities
spinal bifida
avian stem cells in embryos
Is the coming
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Egg
•A typical egg white contains 3.5-4.0
grams of protein
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Egg
recombinant protein could yield up to a
gram or more of the protein in the
naturally sterile egg
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Protein of therapeutic
significance
•interferon beta-1a, in the whites of
eggs laid by transgenic hens using the
employs Lent Vector. Interferon-
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lent virus
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Lentivirus
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lentivirus
•lent viral vectors have the advantage
of infecting both dividing and non-
dividing cells.
•However, they retain stable
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lentivirus
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lentivirus
Lentivirus can be pseudo typed with
ease to infect certain tissues and cell
lines
with greater efficiency. Made to accommodate expression
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lentivirus
•these viral vectors do not elicit
immune responsesin vivo
can be concentrated to titers of 10.
109 Cfu/ ml
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difficulty
•embryos contain about 50,000 cells
before the egg is laid
inserting DNA into just one cell.
gene transfer in other mammals involves
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advantages
•chicken's generation time is around 6
months
shorter than the generation time of a large mammal
, such as a goat, which requires 18 months
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Advantage of egg white
•eggs a hen lays annually 330 eggs
contains
•6.5grams of various proteins
advantage of producing pharmaceuticals in eggs is that egg
Is
simpler
Than
milk
milk
Egg
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Egg white advantages
methods for easily extracting various
proteins from eggs.
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transgenic
Other important fields
neurobiology model
cardiovascular biology
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transgenic
•How can perform
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The MOUSE Life span: approx. 2.5 years
Gestation : 21 days
Litter size: 8 to 12
Generation time: three months
Several inbred and outbreed strains
Genomic database
Most advance genetic technologies
Cost per mouse 5 E p
Housing cost
Over 90% identical to human genome
Large enough for physiological studies
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Mouse
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NON-TRANSGENIC
TRANSGENIC
No modifications to the genome.
Modifications to the genome.
Germ line mutations Somatic cell mutations
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Special facilities
•Specialized glassware
Pulled, Holding and Transfer pipettes are used
for manipulating and moving embryos around.All are
handmade from stock glass tubing
Injection pipettes are machine pulled.
•Microscopes - Surgical, Dissecting, & Inverted
•Micromanipulators – Convert gross hand movements
into micro-movements
•Microsurgical instruments – Used for harvest and
re-implantation of embryos
•Incubator – Used when culturing embryos is required
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Labeled cages
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micro injector
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Microinjection Set-Up
Micromanipulator
Inverted Microscope
Hamilton Syringe
for operating
Holding Pipet Foot Pedal for operating
Microinjection Pipet
Nitrogen
Nitrogen Powered
Microinjection
Apparatus
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Pipets for Pronuclear Microinjection
Internal Diameter
120 m – 180 m
Internal Diameter
55 m – 70 m
Internal Diameter
120 m – 180 m
Pulled Holding Injection Transfer
Internal Diameter
1 m – 3 m
Next
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Surgical instrument
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Surgical instrument
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surgical
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weigh
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Cabinet
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cabinet
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equipment
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Experimental Outline
Procedures begin 3 days before any
embryos are harvested or injected
Next
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Surgical
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handling
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•Pregnant mare’s serum (PMSG)
is administered
intraperitoneally (I.P.) to donor
females
•PMSG mimics follicle
stimulating hormone and
induces the maturation of
oocytes in the ovary
Superovulation
Part I
Next
Day -3
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super ovulation
•Super ovulate donor females to
maximize embryo yield
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Day -1
•Human Chrionic Gonadotropin
(HCG) is administered I.P. to donor
females
•HGC mimics Latinizing hormone
and stimulates the ovulation of the
mature oocytes from the ovary
Superovulation
Part II
Next
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•Set-up mating of donor male and
female mice and recipient
females with vasectomized males
Donor females are then
placed with donor males
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harvest
•Harvest embryos from donor
females
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Embryo Harvest
An abdominal
incision is made and
the ovaries, oviducts
and uteri are
excised
Next
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Ovary Fat Pad
Oviduct
Uterus
Isolating the Oviduct
The structures of the excised portion of the
reproductive tract are identified and the mesentery
torn away to facilitate isolation of the oviduct
Mesentery
Next
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Isolating the Oviduct
The oviduct, along with a small portion
of the uterus is isolated and removed
Next
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Fat pad
Oviduct
Freeing Embryos from Oviduct
Cumulus MassCumulus Mass
The cumulus mass is visualized through the wall of the oviduct
and a needle is used to tear it open and free the embryos
Next
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Media with hyaluronidase
Wash and Grade Embryos
The cumulus mass is placed in a large drop of medium with
hyalurdonidanse. After the cumulus cells have fallen away, the embryos
are transferred through three small rinse drops and graded for quality. Only
embryos suitable for injection advance to the final drop.
Rinse Drops
Embryos for injection
Next
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Grading Embryos
•Healthy, fertilized embryos must be
separated from degenerate and unfertile
embryos for injection
Next
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Zona Pellucida
Embryo
Pronuclear
Polar Bodies
Basic Embryo Anatomy
Next
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Examples of Embryos
Good
Unfertilized
Cleaved
Degenerate
Next
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Photograph showing
examples of good and
degenerate embryos
Next
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Embryos in Injection Dish
Acrylic Frame Glass Cover Slip
Embryo injections are performed on a special microscope slide
composed of an acrylic frame with a glass cover slip attached.
Embryos are placed in the center of a drop of medium that is
covered with oil to prevent movement and dehydration of the drop.
Next
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grading
•Characteristics of a good embryo:
–2 visible pronuclear
–Embryo should fill the zone
pellucida
–Well defined embryo within the
zona pellucida
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injection
•Inject embryos with DNA
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implantation
•Surgically re-implant injected
embryos into pseudo pregnant
recipients
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Pronuclear Microinjection
Using gentle suction, the embryos are held in place by the
holding pipet so that the pronucleus can be injected
Holding Pipet
Next
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Embryo Transfer
• After all embryos have been injected they are inspected and the number of embryos that still appear healthy are
separated for injection. Embryos which did not survive the injection process are discarded
• Using a remote mouth pipe ting device, embryos are loaded into a transfer pipette for re-implantation into
pseudopregnant recipients
• 20-30 embryos will be transferred into one oviduct of each recipient
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Loading the Transfer Pipet
Mineral Oil Air
Embryos in media
Alternate Transfer Pipet Design
The transfer pipette is filled with medium then loaded with the
embryos to be re-implanted in oviduct of the recipient female
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An incision is made in the flank of an
anesthetized mouse
The ovary and oviduct are
then gently pulled through
the incision. A tear is made
in the bursa and the
infundibulum exposed.
The tip of the loaded
transfer pipette is placed
inside the infundibulum and
the embryos gently blown
into the oviduct
The tract is returned to the
abdomen and the incision
site is closed.
Oviduct Transfer

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Transgenic animal secrets

  • 2. you are excellent Mohamed Taha 2 Model for diseases can produce valuable pharmaceuticals information
  • 3. you are excellent Mohamed Taha 3 can be defined as modification of the genetic of an organism through
  • 4. you are excellent Mohamed Taha 4 European Laboratory Animal Associations as an animal in which there has been a deliberate modification of its,the genetic there has been a deliberate modification of its genome
  • 5. you are excellent Mohamed Taha 5 WHY? Study gene function and regulation Genome mapping
  • 6. you are excellent Mohamed Taha 6 • Test knock in gene Knock out gene
  • 7. you are excellent Mohamed Taha 7 Cancer research Transposons transferred from galley fish
  • 8. you are excellent Mohamed Taha 8 •The genes can be modified to jump wily-nilly around cells. By putting them into a batch of identify which genes or combinations of genes lead to cancer.
  • 9. you are excellent Mohamed Taha 9 Jumping genes from jelly fish •By putting them into a batch of human or mouse cells, and watching to see which ones become cancerous
  • 11. you are excellent Mohamed Taha 11 have been made using animals medical advances over the last century
  • 12. you are excellent Mohamed Taha 12 Biomedical research a treatments before attempting to diagnose model of a disease to learn about it and test
  • 13. you are excellent Mohamed Taha 13 Benefits of transgenic? Phytase enzyme poultry animal production
  • 14. you are excellent Mohamed Taha 14 salmon •Improved gain efficiency and protein production Super-salmon
  • 15. you are excellent Mohamed Taha 15 salmon Antifreeze protein in flounder + Chinook salmon
  • 17. you are excellent Mohamed Taha 17 Totipotentcy
  • 18. you are excellent Mohamed Taha 18 Embryonic Stem Cell Lines Isolate inner cell mass (destroys embryo) Heart muscleKidney Liver “Special sauce” (largely unknown) Day 5-6 Blastocyst Inner cells (forms fetus) Outer cells (forms placenta) Heart repaired Culture cells
  • 22. you are excellent Mohamed Taha 22 Nuclear Transfer Lamb 6LL3 (‘Dolly’) Donor: (adult) mammary gland cell - Finn Dorset ewe Recipient: enucleated oocyte - Scottish Blackface ewe
  • 23. you are excellent Mohamed Taha 23 DNA vaccine
  • 24. you are excellent Mohamed Taha 24 EX-Vivo Trans genesis
  • 26. you are excellent Mohamed Taha 26 •1981: First transgenic mouse –Insertion of hGH into a mouse (Singleton, 1999) –Production of monoclonal antibodies & anti-inflammatory agents
  • 27. you are excellent Mohamed Taha 27 CAST, 2003 has been the most common method
  • 28. you are excellent Mohamed Taha 28 2. An animal is given hormonal treatment to produce a large number . Of embryos, and the embryos are collected from the oviduct. 3. The human gene is inserted into the fertilized egg via microinjection 4. The transgenic embryo is placed in a surrogate host which gives Birth to the transgenic animal 5. The offspring is tested for the new gene 1. A human gene responsible for producing a desired protein Is isolated in a laboratory
  • 29. you are excellent Mohamed Taha 29 transgenic •gene transfer is the production of transgenic dairy farm
  • 30. you are excellent Mohamed Taha 30PEW Initiative, 2005 FDA, 2005 Mastitis Disease Resistance Annie, born March 3, 2000, is a clone of a pure-bred Jersey calf genes for Staphylococcus aurousbacteria producing lysostaphin, a protein that kills Staphylococcus aurousbacteria
  • 31. you are excellent Mohamed Taha 31 Lysozyme is a protein found in the tears gene for an antibacterial enzyme found in human breast milk, Lysozyme inhibits the growth of bacteria by destroying the bacterial cell wall Treatment infant diarrhea produce human lysozyme in their milk
  • 32. you are excellent Mohamed Taha 32 Transgenic goat milk contains a protein that could be extracted to make a drug for coronary bypass patients. now in human clinical trials. Millie is aHer The protein,, is called anti-thrombin III goat. transgenic
  • 33. you are excellent Mohamed Taha 33 Steps •the gene that produces AT III is sequenced,. build a synthetic gene and make many copies and
  • 34. you are excellent Mohamed Taha 34 then •The synthetic gene is then attached to the gene forcasein,which acts as apromoter gene.This ensemble is then injected into a. newly fertilized egg
  • 35. you are excellent Mohamed Taha 35 then •. During the first few critical cell divisions, the gene may become attached to the goat's DNA.
  • 36. you are excellent Mohamed Taha 36 then •If successful, the new gene is now a transgenic which will become incorporated into all cells during subsequent divisions
  • 37. you are excellent Mohamed Taha 37 then •The embryo is then transferred to a surrogate mother
  • 38. you are excellent Mohamed Taha 38 Off spring
  • 39. you are excellent Mohamed Taha 39 •Cloning through the ages: –1952: Cloning via nuclear transfer (frog) –1989-1990: First mammals cloned –1995: First cloning via cultured mammalian cells –1997: First cloning via adult cells – DOLLY
  • 40. you are excellent Mohamed Taha 40 history •First cloning via transgenic adult cells – POLLY –1998 – 2000: Cloning of cattle, pigs, mice, goats and monkeys using adult cells –2001: First reported cloned human embryo –2002: First cloned pet
  • 41. you are excellent Mohamed Taha 41 How many genes are there E. coli 4.6 Mb 4,288 genes S. cerevisiae 13.5 Mb 6,034 genes D. melanogaster 165 Mb 12,000 genes C. elegans 97 Mb 19,099 genes H. Sapiens 3,300 Mb 40,000 genes Phenotypes
  • 42. you are excellent Mohamed Taha 42 Which of which •Me can be genetically modified No N o No
  • 44. you are excellent Mohamed Taha 44 Ethics •Generally scientist must be the most honest persons •They know •This is a blessing of Allah
  • 45. you are excellent Mohamed Taha 45 In human •Research on human embryo •Is illegal •Some done it behind
  • 47. you are excellent Mohamed Taha 47 I mean •Animal model for disease •Bioreactor for producing •Therapeutic in milk of •Goat •rabbit •Cow
  • 48. you are excellent Mohamed Taha 48 Transgenic animals are costly: 20,000-30,000 for one animal, and low chances of success. If successful, one animal can produce in its life time 200,000- 300,000 million worth of drugs. A herd of 600 transgenic cows could supply the worldwide demand of human serum albumin (used in the treatment of burns and traumatic injuries)
  • 49. you are excellent Mohamed Taha 49 Mention what is in your list? •Transgenic •Transgenic mouse •Gene expression regulation •Structure function •Cause and effect •In path physiological process
  • 50. you are excellent Mohamed Taha 50 Mention what are in your list •Transgenic mouse •Transgenic rat •Transgenic rabbit •Transgenic goat •Transgenic chicken •Transgenic cow •Transgenic chicken Transgenic quail
  • 51. you are excellent Mohamed Taha 51 Transgenic rabbit •used as model for cardiovascular diseases •Used as a model for AIDS •Used as model for cancer
  • 52. you are excellent Mohamed Taha 52 Transgenic rabbit •The recombinant protein can be produced •low cost •Wide scale
  • 53. you are excellent Mohamed Taha 53 What rabbit milk ? •What is can be in rabbit milk •One of the most attractive protein •Is alfa glycosidase
  • 54. you are excellent Mohamed Taha 54 Rabbit both bioreactor
  • 55. you are excellent Mohamed Taha 55 when multiple preferred analysis be performed on single sample blood chronic studies long term monitoring
  • 56. you are excellent Mohamed Taha 56 Transgenic chicken •to understand normal and abnormal embryo development limb deformities spinal bifida avian stem cells in embryos Is the coming
  • 58. you are excellent Mohamed Taha 58 Egg •A typical egg white contains 3.5-4.0 grams of protein
  • 59. you are excellent Mohamed Taha 59 Egg recombinant protein could yield up to a gram or more of the protein in the naturally sterile egg
  • 60. you are excellent Mohamed Taha 60 Protein of therapeutic significance •interferon beta-1a, in the whites of eggs laid by transgenic hens using the employs Lent Vector. Interferon-
  • 61. you are excellent Mohamed Taha 61 lent virus
  • 62. you are excellent Mohamed Taha 62 Lentivirus
  • 63. you are excellent Mohamed Taha 63 lentivirus •lent viral vectors have the advantage of infecting both dividing and non- dividing cells. •However, they retain stable
  • 64. you are excellent Mohamed Taha 64 lentivirus
  • 65. you are excellent Mohamed Taha 65 lentivirus Lentivirus can be pseudo typed with ease to infect certain tissues and cell lines with greater efficiency. Made to accommodate expression
  • 66. you are excellent Mohamed Taha 66 lentivirus •these viral vectors do not elicit immune responsesin vivo can be concentrated to titers of 10. 109 Cfu/ ml
  • 67. you are excellent Mohamed Taha 67 difficulty •embryos contain about 50,000 cells before the egg is laid inserting DNA into just one cell. gene transfer in other mammals involves
  • 68. you are excellent Mohamed Taha 68 advantages •chicken's generation time is around 6 months shorter than the generation time of a large mammal , such as a goat, which requires 18 months
  • 69. you are excellent Mohamed Taha 69 Advantage of egg white •eggs a hen lays annually 330 eggs contains •6.5grams of various proteins advantage of producing pharmaceuticals in eggs is that egg Is simpler Than milk milk Egg
  • 70. you are excellent Mohamed Taha 70 Egg white advantages methods for easily extracting various proteins from eggs.
  • 71. you are excellent Mohamed Taha 71 transgenic Other important fields neurobiology model cardiovascular biology
  • 72. you are excellent Mohamed Taha 72 transgenic •How can perform
  • 74. you are excellent Mohamed Taha 74 The MOUSE Life span: approx. 2.5 years Gestation : 21 days Litter size: 8 to 12 Generation time: three months Several inbred and outbreed strains Genomic database Most advance genetic technologies Cost per mouse 5 E p Housing cost Over 90% identical to human genome Large enough for physiological studies
  • 75. you are excellent Mohamed Taha 75 Mouse
  • 77. you are excellent Mohamed Taha 77 NON-TRANSGENIC TRANSGENIC No modifications to the genome. Modifications to the genome. Germ line mutations Somatic cell mutations
  • 78. you are excellent Mohamed Taha 78 Special facilities •Specialized glassware Pulled, Holding and Transfer pipettes are used for manipulating and moving embryos around.All are handmade from stock glass tubing Injection pipettes are machine pulled. •Microscopes - Surgical, Dissecting, & Inverted •Micromanipulators – Convert gross hand movements into micro-movements •Microsurgical instruments – Used for harvest and re-implantation of embryos •Incubator – Used when culturing embryos is required
  • 79. you are excellent Mohamed Taha 79 Labeled cages
  • 80. you are excellent Mohamed Taha 80 micro injector
  • 81. you are excellent Mohamed Taha 81 Microinjection Set-Up Micromanipulator Inverted Microscope Hamilton Syringe for operating Holding Pipet Foot Pedal for operating Microinjection Pipet Nitrogen Nitrogen Powered Microinjection Apparatus
  • 82. you are excellent Mohamed Taha 82 Pipets for Pronuclear Microinjection Internal Diameter 120 m – 180 m Internal Diameter 55 m – 70 m Internal Diameter 120 m – 180 m Pulled Holding Injection Transfer Internal Diameter 1 m – 3 m Next
  • 83. you are excellent Mohamed Taha 83 Surgical instrument
  • 84. you are excellent Mohamed Taha 84 Surgical instrument
  • 85. you are excellent Mohamed Taha 85 surgical
  • 86. you are excellent Mohamed Taha 86 weigh
  • 87. you are excellent Mohamed Taha 87 Cabinet
  • 88. you are excellent Mohamed Taha 88 cabinet
  • 89. you are excellent Mohamed Taha 89 equipment
  • 90. you are excellent Mohamed Taha 90 Experimental Outline Procedures begin 3 days before any embryos are harvested or injected Next
  • 91. you are excellent Mohamed Taha 91 Surgical
  • 92. you are excellent Mohamed Taha 92 handling
  • 93. you are excellent Mohamed Taha 93 •Pregnant mare’s serum (PMSG) is administered intraperitoneally (I.P.) to donor females •PMSG mimics follicle stimulating hormone and induces the maturation of oocytes in the ovary Superovulation Part I Next Day -3
  • 94. you are excellent Mohamed Taha 94 super ovulation •Super ovulate donor females to maximize embryo yield
  • 95. you are excellent Mohamed Taha 95 Day -1 •Human Chrionic Gonadotropin (HCG) is administered I.P. to donor females •HGC mimics Latinizing hormone and stimulates the ovulation of the mature oocytes from the ovary Superovulation Part II Next
  • 96. you are excellent Mohamed Taha 96 •Set-up mating of donor male and female mice and recipient females with vasectomized males Donor females are then placed with donor males
  • 97. you are excellent Mohamed Taha 97 harvest •Harvest embryos from donor females
  • 98. you are excellent Mohamed Taha 98 Embryo Harvest An abdominal incision is made and the ovaries, oviducts and uteri are excised Next
  • 99. you are excellent Mohamed Taha 99 Ovary Fat Pad Oviduct Uterus Isolating the Oviduct The structures of the excised portion of the reproductive tract are identified and the mesentery torn away to facilitate isolation of the oviduct Mesentery Next
  • 100. you are excellent Mohamed Taha 100 Isolating the Oviduct The oviduct, along with a small portion of the uterus is isolated and removed Next
  • 101. you are excellent Mohamed Taha 101 Fat pad Oviduct Freeing Embryos from Oviduct Cumulus MassCumulus Mass The cumulus mass is visualized through the wall of the oviduct and a needle is used to tear it open and free the embryos Next
  • 102. you are excellent Mohamed Taha 102 Media with hyaluronidase Wash and Grade Embryos The cumulus mass is placed in a large drop of medium with hyalurdonidanse. After the cumulus cells have fallen away, the embryos are transferred through three small rinse drops and graded for quality. Only embryos suitable for injection advance to the final drop. Rinse Drops Embryos for injection Next
  • 103. you are excellent Mohamed Taha 103 Grading Embryos •Healthy, fertilized embryos must be separated from degenerate and unfertile embryos for injection Next
  • 104. you are excellent Mohamed Taha 104 Zona Pellucida Embryo Pronuclear Polar Bodies Basic Embryo Anatomy Next
  • 105. you are excellent Mohamed Taha 105 Examples of Embryos Good Unfertilized Cleaved Degenerate Next
  • 106. you are excellent Mohamed Taha 106 Photograph showing examples of good and degenerate embryos Next
  • 107. you are excellent Mohamed Taha 107 Embryos in Injection Dish Acrylic Frame Glass Cover Slip Embryo injections are performed on a special microscope slide composed of an acrylic frame with a glass cover slip attached. Embryos are placed in the center of a drop of medium that is covered with oil to prevent movement and dehydration of the drop. Next
  • 108. you are excellent Mohamed Taha 108 grading •Characteristics of a good embryo: –2 visible pronuclear –Embryo should fill the zone pellucida –Well defined embryo within the zona pellucida
  • 109. you are excellent Mohamed Taha 109 injection •Inject embryos with DNA
  • 110. you are excellent Mohamed Taha 110 implantation •Surgically re-implant injected embryos into pseudo pregnant recipients
  • 111. you are excellent Mohamed Taha 111 Pronuclear Microinjection Using gentle suction, the embryos are held in place by the holding pipet so that the pronucleus can be injected Holding Pipet Next
  • 113. you are excellent Mohamed Taha 113 Embryo Transfer • After all embryos have been injected they are inspected and the number of embryos that still appear healthy are separated for injection. Embryos which did not survive the injection process are discarded • Using a remote mouth pipe ting device, embryos are loaded into a transfer pipette for re-implantation into pseudopregnant recipients • 20-30 embryos will be transferred into one oviduct of each recipient
  • 114. you are excellent Mohamed Taha 114 Loading the Transfer Pipet Mineral Oil Air Embryos in media Alternate Transfer Pipet Design The transfer pipette is filled with medium then loaded with the embryos to be re-implanted in oviduct of the recipient female
  • 115. you are excellent Mohamed Taha 115 An incision is made in the flank of an anesthetized mouse The ovary and oviduct are then gently pulled through the incision. A tear is made in the bursa and the infundibulum exposed. The tip of the loaded transfer pipette is placed inside the infundibulum and the embryos gently blown into the oviduct The tract is returned to the abdomen and the incision site is closed. Oviduct Transfer