Transcript TRANSGENIC ANIMALS

TRANSGENIC
ANIMALS
TRANSGENIC ANIMALS-:
INTRODUCTION
OBJECTIVES OF GENE TRANSFER
VECTORS
Methods of modified dna insertion
Transgenic animals
INTRODUCTION
 A TRANSGENIC ANIMAL CONTAINS IN ITS GENOME OR GENES INTRODUCED BY ONE OR THE
OTHER TECHNIQUES OF TRANSFECTION.
 THE GENE INTRODUCED BY TRANSFECTION IS CALLED TRANSGENE.
 IN ANIMALS, TRANSFECTION SPECIFIES THE INTRODUCTION OF A DNA SEGMENT, EITHER
NAKED OR INTERGRATED INTO A VECTOR,INTO AN ANIMAL CELL.
 THE SAME PHENOMENON IS KNOWN AS TRANSFORMATION IN IN ALL OTHER ORGANISMS.

TRANFRMATION HAS L BEEN USED TO DESCRIBE THE CHANGE OF NORMAL TO TUMUOUR
LIKE CELLS.

TRANSGENIC ORGANISMS ARE ABLE TO EXPRESS FOREIGN GENES BECAUSE THE GENETIC
CODE IS SIMILAR FOR ALL ORGANISMS.

THIS MEANS THAT A SPECIFIC DNA SEQUENCE WILL CODE FOR THE SAME PROTIEN IN ALL
ORGANISMS.

THE MOST COMMLY USED TRANSGENIC ANIMAL IS MICE. THE MICE IS ABLE TO PRODUCE
THE HUMAN PROTIEN TO TREAT BLOOD CLOTS.
OBJECTIVES OF GENE TRANSFER
 AIM AT STUDIES ON PROMOTER FUNCTION,REPORTER GENE EXPRESSION,REGULATION OF GENE
EXPRESSION.
 TO OBTAIN THE ESSENTIAL PROTEIN.
 AIMED AT IMPROVING THEIR MILK, MEAT,WOOL PRODUCTION.
 GENES TRANSFERRED IN MILK , URINE OR BLOOD OF ANIMALS , SUCH ANIMALS ARE CALLED BIOREACTORS
AND APPROACH IS MOLECULAR FARMING OR GENE FARMING.
 TO ELEMINATE THE SYMPTOMS OF GENETIC DISEASE AND GENE THERAPY.
 SPECIFIC TRANSGENIC ANIMAL STRAINS ARE CREATED TO FULFIL SPECIALISED EXPPERIMENTAL OR
BIOLOGICAL NEEDS.
A FISH VECTOR-:
 A TYPICAL FISH VECTOR IS A PLAMID e. g . pRSV .
 CONTAIN A SELECTABLE MARKER,(AMPICILLIN RESISTANCE AND THE ORIGIN OF
REPLICATION FROM E. COLI PLASMID pBR322.
 AN ENHANCER/PROMOTER SEQUENCE.
 A MULTIPLE CLONING SITE FOR INSERTION OF THE DNA INSERT .
 A TERMINATION SITE INCLUDING THE POLYADENYLATION SITE.
P ELEMENT VECTORS-:
 DROSOPHILA P ELEMENT HAVE BEEN DEVELOPED AS VALUABLE VECTORS FOR THIS
INVALUABLE GENETIC MATERIAL.
 P ELEMENTS ARE TRANSPOSONS OF 2.9 kb.
 CONTAIN 3 GENES FLANKED BY SHORT (31 bp) INVERTED REPEAT SEQUENCE AT THEIR
ENDS .
 THE GENES ENCODE TRANSPOSASE, WHICH RECOGNISE THE TERMINAL REPEATS AND EFFECTS
TRANSPOSITION OF THE P ELEMENT. A P ELEMENT CLONING VECTOR IS ESSENTIALLY A BACTERIAL
PLASMID VECTOR . E.g. Puc8,
 IT CONTAIN TWO P ELEMENTS. ONE OF THESE P ELEMENTS HAS BOTH ITS TERMINAL REPEATS DELETED SO
THAT IT CAN NO LONGER BE RECOGNISED BY TRANSPOSASE.
 THE OTHER P ELEMENT HAS ITS TERMINAL REPEATS INTACT AND IS ABLE TO TRANSPOSE; THE DNA INSERT
IS PLACED WITHIN THE TRANSPOSASE GENE OF THIS ELEMENT.
SV40 VECTOR-:
• Spherical virus with circular or ds 5243 bp chromosomes, encodes 5 proteinssmall-T,large-T,VP1,VP2,VP3.
• Large –T is essential for viral replication.
• VP1, VP2, VP3 Form viral capsid.
• SV40 developed into 3 types of vectorI. Transducing vector
II. Plasmid vector
III. Transforming vector.
METHODS OF MODIFIED DNA
INSERTION-:
LIPOFECTION
DEAD-DEXTRAN MEDIATED TRANSFECTION
FUSION WITH BACTERIAL PROTOPLAST
RETROVIRAL INFECTION
MICROINJECTION
LIPOFECTION
 DELIVERY OF DNA USING LIPOFECTION.
 LIPOSOMES ARE SMALL VESICLES PREPARED FROM
SUITABLE LIPID.
 CATIONIC LIPIDS ARE USED FOR LIPOSOMES BECAUSE DNA
SPONTANEOUSLY AND EFFICIENTLY COMPLEXES WITH
THESE LIPOSOMES.
 THE CATIONIC LIPOSOMES HAVE A SINGLELIPID BILAYER
MEMBRANE AND THEY BIND TO CELLS EFFICIENTLY
MICROINJECTION
 DNA SOLUTION IS INJECTED DIRECTLY INTO THE NUCLEUS OF A CELL.
 THE MALE PRONUCLEUS IS CHOSEN FOR MICROINJECTION BECAUSE IT IS
MUCH LARGER THAN THE FEMALE PRONUCLEUS OF FERTILISED
MAMMALIAN OVA.
 FEMALE MICE ARE SUBJECTED TO A REGIME OF PREGANT MARE SERUM
GONADOTROPHIN(PMSG).
 IT STIMULATES GROWTH AND DEVELOPMENT OF FOLLICLES , CONTAINS
OOCYTES.
FUSION WITH BACTERIAL
PROTOPLASTS
• PROTOPLASTS ARE PREPARED FROM BACTERIAL CELLS THAT
CONTAIN THE EXOGENOUS DNA(THE DNA FRAGMENT TO BE
TRANSFERRED IN THE CULTURED CELLS).
• RECOMBINANT DNA WAS CLONED IN BACTERIAL CELLS.
• PROTOPLAST IS FUSED USING PEG.
ELECTROPORATION
• IN THIS METHOD TRANFECTION MIXTURE IS EXPOSED FOR A VERY BRIEF
PERIOD TO A VERY HIGH VOLTAGE GRADIENT.
• TREATMENT OF CELLS WITH COLCEMID BEFORE ELECTROPORATION INCREASE
FREQUENCY OF TEANSFECTION.
• THIS IS DUE TO ARREST OF CELLS AT METAPHASE .
• LINEAR DNA IS MORE EFFICIENT THAN CIRCULAR DNA.
AIM FOR PRODUCTION OF
TRANSGENIC ANIMALS
• Production of larger number of eggs either naturally.
• Short breeding cycle i.e. time taken from birth to reaching the reproductive age.
• It is desirable that ovulation occurs throughout the year so that ova are readily
available for experiment.
• The size and the structure of eggs should be amenable to micro injection.
• Production and maintenance of embryonic stem cell lines capable of giving rise to
germ cells is essential for the application of ES cell transfer technology.
TRANSGENIC ANIMALS
1. TRANSGENIC MICE-:
• Mouse is the most preferred mammal for studies on gene transfer due to its
many favorable features like oestrus cycle and gestation period ,relatively
short generation time , convenient in vitro fertilization.
• As a result , the techniques for gene transfer and transgenic production
have been developed using mice as models; subsequently , these approaches
have been modified to adapt them to other animal species.
TRANSGENIC RABBITS-:
o Rabbits are quite promising for gene farming or molecular farming, which aims at the production
of recoverable quantities of biologically important proteins encoded by the transgenes.
o Transgenic animals used for this purpose are popularly called bioreactors.
o These transgenes are expressed in mammary tissue so that their protein product are secreted in milk..
o The human genes encoding valuable proteins transferred into rabbits; interleukin 2, growth hormone,
tissue plasminogen activator , ά1 antitrypsin etc.
TRANSGENIC CATTLE-:
o THE ONLY SUCCESSFUL TRANFECTION TECHNIQUE IN CATTLE IS MICROINJECTION OF
FERTILISED OVA.
o THE CHIEF OBJECTIVES OF TRANSGENIC PRODUCTION IS -:
i.
INCREASED MILK OR MEAT PRODUCTION.
ii.
MOLECULAR FARMING.
iii.
IMPROVED PROPERTIES AND PROPORTIONS OF CASEIN, CONTENT OF LACTOSE AND
BUTERFAT IN MILK.
iv.
ENHANCE RESISITANCE TO VIRAL AND BACTERIAL PATHOGEN.
TRANSGENIC GOAT AND FISH-:
 GOAT-:
GOATS ARE BEING USE AS BIOREACTORS.
HUMAN GENES HAVE BEEN INTRODUCED IN GOATS AND THEIR EXPRESSION ACHIEVED
IN MAMMARY TISSUE.
 FISH -:
GENE TRANSFER HAVE BEEN SUCCESSFUL IN SEVERAL FISH.
THE GENES TRANFERRED ARE-: growth
hormone, chicken δ-crystalline protein,winter flounder
antifreeze protein, E.coli β – galactosidase.
TRANSFECTION IS ACHIVED BY MICROINJECTION OF DNA .
TRANSGENIC SHEEP-:
 TRANSGENIC SHEEP IS PRODUCED TO ACIEVE BETTER GROWTH , MEAT PRODUCTION AS
WELL AS TO SERVE AS BIOREACTOR.
 HUMAN GENES ARE ALSO EXPRESSED IN SHEEP.
 INCREASED WOOL PRODUCTION AND IMPROVED WOOL QUALITY ARE IMPORTANT
OBJECTIVES OF TRANSGENIC SHEEP.