transgenic animal secrets
DESCRIPTION
Modification n animal genome transgenic animal useful fr get some valuable therapeutics model animals human being trans genesis is illegal but some don't respectTRANSCRIPT
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Model for diseasescan produce valuable
pharmaceuticalsinformation
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can be defined as modification of the genetic
of an organismthrough
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European Laboratory Animal Associations
as an animal in which there has been a deliberate modification of its ,the
geneticthere has been a deliberate modification of its
genome
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WHY?
Study gene function and regulationGenome 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
productionSuper-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 5-6Blastocyst
Inner cells(forms fetus)
Outer cells(forms placenta)
Heartrepaired
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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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
microinjection4. The transgenic embryo is placed in a surrogate host which gives
Birth to the transgenic animal5. 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 ResistanceAnnie, 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 wallTreatment 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
demandof 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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Rabbitboth
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 embryosIs the coming
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Egg•A typical egg white contains 3.5-4.0
grams of protein
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Eggrecombinant 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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lentivirusLentivirus 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 eggIs
simplerThanmilk
milkEgg
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Egg white advantagesmethods 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
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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
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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
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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
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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
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Ovary Fat Pad
Oviduct
Uterus
Isolating the OviductThe structures of the excised portion of the
reproductive tract are identified and the mesentery
torn away to facilitate isolation of the oviduct
Mesentery
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Isolating the Oviduct
The oviduct, along with a small portion
of the uterus is isolated and removed
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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
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Media with hyaluronidase
Wash and Grade EmbryosThe 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
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Grading Embryos
•Healthy, fertilized embryos must be separated from degenerate and unfertile
embryos for injection
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Zona Pellucida
Embryo
Pronuclear
Polar Bodies
Basic Embryo Anatomy
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Examples of Embryos
Good
Unfertilized
Cleaved
Degenerate
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Photograph showing
examples of good and
degenerate embryos
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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.
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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 MicroinjectionUsing gentle suction, the embryos are held in place by the
holding pipet so that the pronucleus can be injected
Holding Pipet
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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 AirEmbryos 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