CLONINGCLONING

Prepared by: Nurul Faizah binti Jusoh

INTRODUCTION

Production of genetically identical individuals that

have identical nuclear DNA

Cloning

Technologies

Therapeutic cloning

Reproductive cloning

Recombinant DNA technology

Embryo

cloningPreimplantation

DNA cloning

Molecular cloning

Gene cloning

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

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)

Stem Cells

� Bone marrow transplantation

� Replacement cells to treat

� Heart disease

Alzheimer's� Alzheimer's

� Cancer

� Diabetes

� Parkinson's disease

� Spinal cord injury

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)

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

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

SCNT

� Sources of somatic cells

� Cell from individual

� Cells grown in culture

� Frozen tissue� Frozen tissue

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)

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

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

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)

Limitation

• Ethical boundaries

• SCNT: still in lab practice

• Case: Dolly died due to bad disease resistant • Case: Dolly died due to bad disease resistant

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

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

How?

DNA+Restriction Enzyme=cut the DNA

DNA Extraction

Ligation

Selection

Transformation

Analysis

Ligation

How?

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

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

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".

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

DNA ligase

� It is a special type of ligase enzyme that create the

phosphodiester bond to fully repair the DNA

Specific palindromic sites

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

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.

Vector: Type & DNA SizeTYPE DNA SIZE (kb)

Plasmid < 10

Bacteriophage ג 9-20

Cosmid 33-47

BAC 75-125

YAC 100-1000

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.

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)

Plasmid: pUC� Multiple cloning sites

� Easier selection – Blue/White Selection

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

Blue/White Selection

� Based on the enzymatic reaction of β-galactosidase.

White

X-gal

α-complementation.

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

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

Analysis� Cloned DNA segments recovered from host cells for

purification and analysis

� Verification by:

� PCR� PCR

� Southern Blot

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

Challenges/Limitation� Challenges on create Restriction Map

� Long term to obtain a gene by cloning

Benefits of

CloningCloning

DNA Cloning - Uses

� Gene therapy

� Genetic engineering of organisms

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

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),

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

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). ).

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.

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.

Limitation

• Ethical boundaries

• SCNT: still in lab practice