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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

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 OviductThe 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 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

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 MicroinjectionUsing 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 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