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CLONINGCLONING
Prepared by: Nurul Faizah binti Jusoh
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INTRODUCTION
Production of genetically identical individuals that
have identical nuclear DNA
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Cloning
Technologies
Therapeutic cloning
Reproductive cloning
Recombinant DNA technology
Embryo
cloningPreimplantation
DNA cloning
Molecular cloning
Gene cloning
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Therapeutic Cloning� “Embryo cloning"
� Used to create cloned embryos for research purposes
� For stem cell research - find methods to treat human � For stem cell research - find methods to treat human
diseases
� Goal
� To harvest stem cells that can be used to study
human development and to treat disease
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Therapeutic Cloning� Stem cells
� Cells have ability to divide and give rise to both
specialized cells and more stem cells
� Derived from � Derived from
� Adults
� Preimplantation embryos (Embryonic stem cells)
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Stem Cells
� Bone marrow transplantation
� Replacement cells to treat
� Heart disease
Alzheimer's� Alzheimer's
� Cancer
� Diabetes
� Parkinson's disease
� Spinal cord injury
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Reproductive Cloning
� A technology used to generate an animal that has same
nuclear DNA as another currently or previously existing
animal
� Dolly the sheep� Dolly the sheep
� How Is Reproductive Cloning Done?
� Somatic cell nuclear transfer (SCNT)
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SCNT
� Starts with removal of polar body and chromosomes from an oocyte
� Enucleated oocyte
� Donor cell then inserted into perivitelline space of � Donor cell then inserted into perivitelline space of enucleated oocyte
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SCNT
� Oocyte and donor cell are fused and activated by an electric pulse to begin cell division
� Developed embryos transferred to surrogate femalesfemales
� Birth of an individual
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SCNT
� Sources of somatic cells
� Cell from individual
� Cells grown in culture
� Frozen tissue� Frozen tissue
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How did scientists clone dolly the sheep?
� The empty unfertilised egg cell gains a new nucleus from the adult body cell.
� The embryo of this cell was
Artificial cloning
1. Nucleus is 2. Nucleus is
taken out of
Unfertilised
egg cell
(ovum)
Body cell
� The embryo of this cell was then grown for a few days.
� This embryo was then implanted into a sheep's womb.
� The new animal was a clone of the animal that donated the nucleus.
1. Nucleus is
taken out of
unfertilised
egg cell
taken out of
body cell
3. Body cell
nucleus is placed
in empty
unfertilised egg
cell.
Unfertilised
egg cell
(ovum)
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Advantages (SCNT)
� Harvesting donor cells from transgenic animal
� Genetic modification of cultured donor cells prior to nuclear transfer
Producing genetically modified cloned offspring� Producing genetically modified cloned offspring
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Genetically Modified Cloned Offspring
� Biotechnological applications
� Production of pharmaceuticals
� Xenotransplantation
� Study and eradication of human disease� Study and eradication of human disease
� Improvement of livestock
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Production Of Pharmaceuticals
� Gene expressing human coagulation factor IX introduced into ovine fetal fibroblasts
� linked to another gene with a high level of expression in mammary glandexpression in mammary gland
� Protein expressed in milk
� Insulin for diabetes
� Interferon for viral infections
� Tissue plasminogen activator (which dissolves blood clots)
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Limitation
• Ethical boundaries• SCNT: still in lab practice• Case: Dolly died due to bad disease resistant • Case: Dolly died due to bad disease resistant
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Recombinant DNA Cloning
• The process of introducing a foreign DNA (or gene) into a host (bacterial, plant or animal) cell
•It is the simple cloning of a gene that his been around since 1970.
• Recombinant DNA is combination of DNAs that has been created artificially (not natural).
•DNA from two or more sources is incorporated into a single recombinant molecule
•Purpose: to simplify isolation and manipulation gene interest via random replication
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rDNA Technology� Procedures by which DNA from different species can be
isolated, cut and spliced together
� New "recombinant " molecules are then multiplied in
quantity in populations of rapidly dividing cells (e.g. quantity in populations of rapidly dividing cells (e.g.
bacteria, yeast).
� Uses methods derived from biochemistry of nucleic acids
coupled with genetic techniques originally used for the
study of bacteria and viruses
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How?
DNA+Restriction Enzyme=cut the DNA
DNA Extraction
Ligation
Selection
Transformation
Analysis
Ligation
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How?
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Steps
Restriction enzymes “cuts” DNA
at specific sites (restriction fragments)
Cut the DNA
DNA ligase “pastes” the DNA
fragments together
The cut segments are inserted into
other DNA molecules that serves as vectors
Transformation
Ligation
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Restriction enzymes� DNA cutting enzymes. Restriction endonucleases
� Cuts DNA at a specific site defined by a sequence of
bases in the DNA (recog.site) forming “sticky ends”
� Palindromic sites� Palindromic sites
� 4-8 bases
� Ex: EcoR1,TaqA1, Sau3A
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Restriction enzymes� DNA cutting enzymes. Restriction endonucleases
� Cuts DNA at a specific site defined by a sequence of
bases in the DNA (recog.site) forming “sticky ends”
� Palindromic sites � Palindromic sites
� 4-8 bases
� Ex: EcoR1, TaqA1 and Sau3A
� EcoR1 will cut at
5’-G AATT C-3’
3’-C TTAA G-5’
word or number that reads the same "left to
right" as it does "right to left".
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Steps
Restriction enzymes “cuts” DNA
at specific sites (restriction fragments)
Cut the DNA
DNA ligase “pastes” the DNA
fragments together
The cut segments are inserted into
other DNA molecules that serves as vectors
Transformation
Ligation
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DNA ligase
� It is a special type of ligase enzyme that create the
phosphodiester bond to fully repair the DNA
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Specific palindromic sites
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StepsRestriction enzymes “cuts” DNA
at specific sites (restriction fragments)
DNA ligase “pastes” the DNA
fragments together
Cut the DNA
Ligation
The cut segments are inserted into
other DNA molecules that serves as vectors
Plasmid + DNA = rPlasmid (DNA Recombinant)
Propagation in host cell (CLONES)
Transformation
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Vector� Carrier DNA molecules = transfers the rDNA into the host
cell.
� Within host cells, vectors can replicate producing many
DNA segments = identical copies (CLONES)DNA segments = identical copies (CLONES)
� Host cells pass the recombinant DNA molecules on their
progeny = population of cells.
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Vector: Type & DNA SizeTYPE DNA SIZE (kb)
Plasmid < 10
Bacteriophage ג 9-20
Cosmid 33-47
BAC 75-125
YAC 100-1000
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Plasmid
� Molecules of DNA that are found in bacteria
� Small (a few thousand base pairs)
� Circular DNA
� Occur naturally in many species of bacteria
� Naturally replicate and are passed on to future
generations of bacterial cells.
� Usually carry only one or a few genes
� Act as a system to transfer genetic material to other
bacteria, allowing those to express the transmitted
genes.
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Plasmid: pBR322� One of the first vectors developed, and the most popular
� 2 sets of antibiotic resistance genes
� Ampicillin Resistance (β-lactamase gene)
Tetracycline Resistance (tet gene)� Tetracycline Resistance (tet gene)
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Plasmid: pUC� Multiple cloning sites
� Easier selection – Blue/White Selection
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StepsRestriction enzymes “cuts” DNA
at specific sites (restriction fragments)
DNA ligase “pastes” the DNA
fragments together
The cut segments are inserted into
Cut the DNA
Ligation
The cut segments are inserted into
other DNA molecules that serves as vectors
Plasmid + DNA = rPlasmid (DNA Recombinant)
Propagation in host cell (CLONES)
Selection by antibiotic
Identify the clones
Transformation
Selection
Analysis
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Blue/White Selection
� Based on the enzymatic reaction of β-galactosidase.
White
X-gal
α-complementation.
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Blue/White SelectionBacteria plus empty
plasmid Non-transformed bacteriaBacteria with
plasmid plus insert
Only colonies
from bacteria that
have plasmid
Nutrient media plus
antibiotic plus X-Gal
Overnight growth
Colonies with insert - white
Colonies w/o insert - blue
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StepsRestriction enzymes “cuts” DNA
at specific sites (restriction fragments)
DNA ligase “pastes” the DNA
fragments together
The cut segments are inserted into
Cut the DNA
Ligation
The cut segments are inserted into
other DNA molecules that serves as vectors
Plasmid + DNA = rPlasmid (DNA Recombinant)
Propagation in host cell (CLONES)
Selection by antibiotic
Identify the clones
Transformation
Selection
Analysis
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Analysis� Cloned DNA segments recovered from host cells for
purification and analysis
� Verification by:
� PCR� PCR
� Southern Blot
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Advantages� Provide a pure sample of a gene
� Forensic applications - All individuals are genetically All individuals are genetically
unique = a distinct "genetic fingerprint“, all types of unique = a distinct "genetic fingerprint“, all types of
specs, old and newspecs, old and newspecs, old and newspecs, old and new
�� Isolate genes that have not been studied beforeIsolate genes that have not been studied before
�� Isolating long genesIsolating long genes
� Genome sequencing
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Challenges/Limitation� Challenges on create Restriction Map
� Long term to obtain a gene by cloning
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Benefits of
CloningCloning
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DNA Cloning - Uses
� Gene therapy
� Genetic engineering of organisms
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Xenotransplantation
� Aim:
To develop animals whose organs will not cause an immunological response and destroy transplanted tissue when transferred to humanswhen transferred to humans
� Pig organs
� Hearts, lungs, kidneys, liver
� Neural tissue for Parkinson's
� Islets cells for diabetes patients
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Medicine
� Medicine –production of industrial and commercial production of industrial and commercial compoundscompounds
�� Insulin Insulin –– DiabetesDiabetes
�� drugs (drugs (angiostationangiostation and and endostatinendostatin) ,) ,�� drugs (drugs (angiostationangiostation and and endostatinendostatin) ,) ,
�� Factor VIII Factor VIII –– HaemophiliaHaemophilia AA
�� Factor IX Factor IX –– HaemophiliaHaemophilia BB
�� EPO EPO –– AnaemiaAnaemia
�� Interleukins and Interleukins and interferonsinterferons
�� Tissue Tissue plasminogenplasminogen activator activator –– dissolve blood clotsdissolve blood clots
�� Hormones = GH, parathyroid Hormones = GH, parathyroid
�� OxytocinOxytocin
�� Adenosine Adenosine deaminasedeaminase ––sevsev. Com. . Com. ImmImm (SCID)(SCID),
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Cont’dCont’d
�� Diagnostic kitsDiagnostic kits –– HepHep, AIDS, AIDS
�� AntibioticsAntibiotics
� genetic testing,––Mapping the chromosomal location of Mapping the chromosomal location of genetic disorders. RFLP, DNA fingerprinting genetic disorders. RFLP, DNA fingerprinting –– the HG the HG projectprojectprojectproject
� Gene therapy – manipulation of DNA to tx diseases by altering individuals genes. (CF, PKU, DMD) ETHICAL ISSUES
� Forensic applications - All individuals are genetically All individuals are genetically unique = a distinct "genetic fingerprint“, all types of unique = a distinct "genetic fingerprint“, all types of specs, old and newspecs, old and new
� Animals = models of genetic diseases models of genetic diseases EgEg GE mice GE mice
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Vaccines
�� Vaccines :Vaccines :vvareare created by transferring the genes that created by transferring the genes that
determine a pathogen's surface configuration to a determine a pathogen's surface configuration to a
microorganism.microorganism.
�� When the GM microorganism is used in a vaccine, its When the GM microorganism is used in a vaccine, its �� When the GM microorganism is used in a vaccine, its When the GM microorganism is used in a vaccine, its
surface stimulates the production of antibodies.surface stimulates the production of antibodies.
�� These antibodies protects an individual against the These antibodies protects an individual against the
pathogen.pathogen.
�� Successful vaccines have been produced for influenza, Successful vaccines have been produced for influenza,
cold sores, and hepatitis B(cold sores, and hepatitis B(HBsAgHBsAg). ).
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Animals
�� Models of GDModels of GD : GA to be vulnerable to a disease so that : GA to be vulnerable to a disease so that
the mechanism of the disease can be investigated and the mechanism of the disease can be investigated and
potential therapeutic agents tested. potential therapeutic agents tested.
�� Eg. mice GA with a cancerEg. mice GA with a cancer--predisposing gene. Research predisposing gene. Research �� Eg. mice GA with a cancerEg. mice GA with a cancer--predisposing gene. Research predisposing gene. Research
to test the consequences of exposure to various potential to test the consequences of exposure to various potential
carcinogens and to test the efficacy of preventive drugs.carcinogens and to test the efficacy of preventive drugs.
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Agriculture
� Animals –– improved quality/quantity of meat productsimproved quality/quantity of meat products
� Plants –– Gene altered plants : improved crops Gene altered plants : improved crops –– quantity, quantity,
quality and pest resistance. quality and pest resistance. EgEg golden rice golden rice –– GM to GM to
contain beta carotene contain beta carotene –– vitvit A deficiency.A deficiency.contain beta carotene contain beta carotene –– vitvit A deficiency.A deficiency.
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Limitation
• Ethical boundaries• SCNT: still in lab practice
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