Jayanti Tokas, MS, PhD 1 ; Rubina Begum, MS. PhD 1 ; Shalini Jain, MS, PhD 2 and Hariom Yadav, MS, PhD 2 1 Department of Biotechnology, JMIT, Radaur 2 NIDDK, National Institutes of Health, Bethesda, MD 20892, USA Corresponding:

Cloning means making an organism identical to another In nature :- Identical Twins Occurs naturally in single celled organisms

Cloning Scientific technique to create genetically identical organisms Production of one or more individual plants or animals(whole/in part) that are genetically identical

Types of Cloning 1.Embryo Cloning 2.Reproductive cloning 3.Therapeutic Cloning

a) Embryo cloning Produce mono zygotic twins or triplets Fertilized embryo remove one/more cells Encourage to develop into embryo Twins /triplets have identical DNA

b) Reproductive cloning Produce a duplicate of existing animal Used to clone sheep & other mammals Produce several genetic defects Medical ethicists- Immoral procedure to be done on humans as it is unsafe & unethical

Newly formed embryo containing DNA from somatic cell cell division implant (Diploid )

(Amy Coxon.,2001)

c) Therapeutic Cloning Somatic Cell Nuclear Transfer(SCNT) Biomedical Cloning Research Cloning Regenerative Medicine Nuclear Transplantation Therapy(NTT)

Therapeutic cloning (research cloning) is when stem cells are extracted to grow into a piece of human tissue which is encouraged to grow into a human organ for transplant

Therapeutic Cloning DNA is extracted from a human’s cell inserted into a woman’s ovum develop and produce stem cells. stem cells are removed from the pre-embryo grown into specific organ transplanted into the patient.

Culturing of pluripotent Stem Cell from fertilized embryo (David Cameron, 2001)

Culturing of pluripotent Stem Cells from cloned embryo (David Cameron, 2001)

Need for Therapeutic Cloning o Implantation of human Embryonic stem (ES) cells is not safe unless they contain patient’s own DNA o Efforts to repair central nervous system (CNS) disorders o Strictly Regulated

Technology behind Therapeutic Cloning Removing nucleus of egg cell Replace with nucleus of a somatic cell Stimulate cell division

Removal of nucleus from adult cell (Irving Weissman,2002)

Transfer of nucleus to enucleated oocyte (Irving Weissman,2002)

Selection of embryonic stem cells

Cloning Techniques 1. Roslin technique 2. Honolulu technique

Isolation of Donor Cell (Ian Wilmut,1995)

Fusion of Donor nucleus to enucleated embryo (Ian Wilmut,1995)

Honolulu technique Developed at University of Hawaii, Honolulu Does not use electric charge for cell fusion Fused cell- given a chemical bath Three donor cells- sertoli, neuronal & cumulus cells

Value of Therapeutic Cloning for patients Potential application in commonly occurring diseases Heart disease Insulin dependent diabetes Parkinson disease Burns Most attractive feature :- Create perfectly matched transplant material

Mitochondrial Disease Mitochondria are component of cells inherited from mother’s egg They carry their own genes & are crucial to fundamental process in all the body cells

Create New ES cells Cells currently available for research are insufficient because:- * Not sufficiently racially or ethnically divers * Scientist need more cell lines to fulfill the promise of stem cell research

Problems associated with Therapeutic Cloning 1.Developing cures 2.Is an embryo a human person ? 3.Stability of stem cells 4.Where would the eggs come from ?

Ethical concern about Therapeutic Cloning 1. Moral status of organisms created by cloning 2.Getting stem cells 3.Getting eggs

Getting Stem Cells Adult organs (morally OK,not technically) Fetal tissue

Getting eggs  Health risk concerning the donors  Paying the donors- commercializing the human reproductive material  Ethical issue related to donors

Therapeutic Cloning is governed by - Human Fertilization and Embryology Authority (1990) Authority is administered by- HFEA which can only license research on embryos up to 14 days old for designate purposes

Create cloned Human cells Use to * better understand human development * provide resources for Stem cell research * develop new treatment for disease Application in genetic disease,serious injury Opportunity to obtain perfectly matched transplant tissue for treating presently incurable condition May lead to development of sophisticated alternatives that do not require eggs, embryonic cells Practical clinical application not expected for at least 10 year

 Public opinion   55% want to ban reproductive cloning but allow TC  30% want to ban both  12% want no ban to both  3% undecided Hence,TC has come in Law (CAMR,March2003)

“To allow cloning would be to move towards a society in which human beings are grown for spare body parts & children are engineered to custom specification – that is not acceptable” I believe all human cloning is wrong ….. any thing other than a total ban on human cloning would be unethical President Bush says on Stem cell policy

1. Treatment for:- Diabetes, Heart diseases,Parkinson's disease, Disease of Bone & cartilage, Immunodeficiency diseases 2. Infertility treatments or to assist couples who know that one parent carries gene for potentially lethal disease 3. Prevent mitochondrial disease 4. Help scientist to understand how stem cell develop 5) Understanding how nuclear reprogramming occurs during CNR would eventually help scientists develop alternative Stem cell therapy techniques that reprogram cells without the need to use eggs, embryos or cloning