Cloning

Dr. Abdelsalam Talafha, DVM

Diplomate, American College of Theriogenologists

Cloning

• Production of genetically identical individuals that have identical nuclear DNA

Cloning Technologies

1. Recombinant DNA technology• DNA cloning• Molecular cloning• Gene cloning

2. Reproductive cloning

3. Therapeutic cloning• Embryo cloning

DNA Cloning

• Transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid

• Plasmids• Self-replicating extra-chromosomal circular

DNA molecules, distinct from normal bacterial genome

DNA Cloning - Uses

• Gene therapy• Genetic engineering of organisms• Genome sequencing

Reproductive Cloning

• A technology used to generate an animal that has same nuclear DNA as another currently or previously existing animal

• Dolly• 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 enucleated oocyte

SCNT

• Oocyte and donor cell are fused and activated by an electric pulse to begin cell division

• Developed embryos transferred to surrogate females

• Birth of an individual

SCNT

• Sources of somatic cells• Cell from individual• Cells grown in culture• Frozen tissue

Therapeutic Cloning

• Production of human embryos for use in research

• 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

• Adults • Preimplantation embryos (Embryonic stem

cells)

Stem Cells

• Replacement cells to treat • Heart disease• Alzheimer's• Cancer• Diabetes• Parkinson's disease• Spinal cord injury

SCNT- Potential Biotechnological Applications

• Harvesting donor cells from transgenic animal• Genetic modification of cultured donor cells

prior to nuclear transfer• Producing genetically modified cloned

offspring

Genetically Modified Cloned Offspring

• Biotechnological applications • Production of pharmaceuticals• Xenotransplantation• 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 gland

• Protein expressed in milk

Production Of Pharmaceuticals

• Insulin for diabetes• Interferon for viral infections• Tissue plasminogen activator (which

dissolves blood clots)

Xenotransplantation

• Aim:• To develop animals whose organs will not

cause an immunological response and destroy transplanted tissue when transferred to humans

Xenotransplantation

• Pig organs• Hearts, lungs, kidneys, liver • Neural tissue for Parkinson's • Islets cells for diabetes patients

Study Human Disease

• Sheep model to investigate human cystic fibrosis

• Cloned sheep used for drug testing and to evaluate new therapies

Improvement Of Livestock

• Cloning animal with excellent traits• Production of a large number of clones from

high quality animals • Allow overall genetic improvement of herd

• Repopulate endangered animals

Animal Cloning

• Sheep, cattle, goats, pigs, and mice• Cloning efforts in rabbits, rats, cats, dogs,

and horses are ongoing

Risks Of Cloning

• Reproductive cloning expensive and highly inefficient

• > 90% of cloning attempts fail to produce viable offspring

Risks Of Cloning

• High rate of fetal loss during pregnancy • Compromised immune function • Higher rates of infection• Tumor growth• Early neonatal death • Abnormally large at birth• Die mysteriously

Risks Of Cloning

• Cloned fetuses have abnormalities• Abnormal placentation• Pregnancy toxemia• Hydroallantois

Should Humans Be Cloned?

• Due to• Inefficiency of animal cloning • Lack of understanding about reproductive

cloning• Risks of cloning

Should Humans Be Cloned?

• Unethical to attempt to clone humans• Same problems would be expected in

human cloning• We do not know how cloning could impact

mental development

Should Humans Be Cloned?

• High risk to health of fetus or infant and mother

• Psychological risks for mother as a result of • Late spontaneous abortions • Birth of a stillborn child • Birth of a child with severe health problems