Unit IV Origin of organism cloning Types of nuclear transfer Cloning procedure in sheep Application of cloning in conservation Difference between therapeutic and reproductive cloning Highlight ethical issues related to cloning Prospects of human cloning

Two vector expression system Directed mutagenesis Transposone mutagenesis Gene targeting Site specific recombination

EBT 501, Genetic Engineering Origins of organismal cloning in developmental biology research on frogs; nuclear transfer procedures and the cloning of sheep (Dolly) & other mammals; applications in conservation; therapeutic vs. reproductive cloning; ethical issues and the prospects for human cloning; Two-vector expression system; two-gene expression vector, Directed mutagenesis; transposon mutagenesis, Gene targeting, Site specific recombination

Nuclear transfer procedures and the cloning of sheep (Dolly) & other mammals

Fertilization vs. Cloning (somatic cell nuclear transfer, SCNT)

Origins of organism cloning in developmental biology research on frogs Life Cycle of the frog

The origin of nuclear transfer started in 1952 when R Briggs and TJ King performed first successful nuclear transfer experiments in frog species Rana pipens (Leopard frog) This was done by transplanting nuclei from the blastula of the frog Rana pipens to an enucleated eggs and obtaining a number of normal embryos. Nuclear transfer is an embryological technique, and involves removal of the nucleus from an egg and replacement with the nucleus of another donor cell. This experiment paved the way for what we know today as Organism cloning

Later, Gurdon 1986 showed that in Xenopus laevis (African clawed frog), nuclei from various types of cell in the swimming tadpole can be transplanted to an egg that has been UV-irradiated to destroy the peripheral chromosomes, and similar results were obtained. The important principle established was that, while animal cells become irreversibly committed to their fate as development proceeds, the nuclei of most cells still retain all the genetic information required for the entire developmental programme and can, under appropriate circumstances, be reprogrammed by the cytoplasm of the egg to recapitulate development Earlier the developmental stage at which nuclei are isolated, the greater their potential to be reprogrammed. This allows animals with specific and desirable traits to be propagated and have applications in farming to

In 1989 Smith & Wilmut demonstrated for the first time the Nuclear transfer in mammals in which donor nuclei were obtained from the morula or blastocyst-stage embryo and transferred to an enucleated egg or oocyte (from which the nucleus had been removed with a pipette) Later on this procedure has been performed successfully for rabbits and other farm animals such as sheep, pigs and cows, which are far more amenable to the process than mice. The donor nucleus can also be introduced by promoting fusion between enucleated egg and a somatic cell by giving a brief electric pulse, as it activates embryonic development by stimulating the mobilization of calcium ions.

In 1995, Campbell and Wilmut produced two live lambs, Megan and Morag by nuclear transfer from cultured embryonic cells. This demonstrated the principle that mammalian nuclear transfer was possible using a cultured cell line In 1996 Campbell and Wilmut reported the birth of Dolly, following nuclear transfer from an adult mammary epithelial cell line in This was the first mammal to be produced by nuclear transfer from a differentiated adult cell, and aroused much debate among both scientists and the public concerning the possibility of human cloning However, the success rate was very low: only one of 250 transfer experiments produced a viable lamb. Similar transfer experiments have since been carried out in mice, cows, pigs and goats

The production of a transgenic mammal by nuclear transfer from a transfected cell line was first achieved by Schnieke in 1997, who introduced the gene for human factor IX into fetal sheep fibroblasts and transferred the nuclei to enucleated eggs. The resulting sheep, Polly, produces the recombinant protein in her milk and can therefore be used as a bioreactor In 2000, McCreath succeeded in producing a transgenic sheep by nuclear transfer from a somatic cell whose genome had been specifically modified by gene targeting. A foreign gene was introduced into the COL1A1 locus and was expressed at high levels in the lamb.

estimates 200,000 clones per year for reproduction 1999 – Richard Seed, M.D. announces plans to develop nuclear transplantation technique for humans estimates 200,000 clones per year for reproduction 2005 – Ian Wilmut group granted license (U.K) for therapeutic cloning of human embryos Problems with nuclear transfer: very low efficiency (1%) high frequency of developmental abnormalities

Difference between therapeutic and reproductive cloning ethical issues related to cloning Prospects of human cloning

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.

What are its uses? - It is used for medical purposes, such as creating organs to transplant into a patient in need of that organ. If replacement organs are available to the sick and dying people, countless numbers of lives could be saved. Therapeutic cloning is a fast and efficient way to ‘repair’ damaged organs. - Therapeutic cloning can be used to make insulin-secreting cells to cure for diabetes; nerve cells to cure stroke or Parkinson’s disease

How is it done? DNA is extracted from a human’s cell. The DNA is inserted into a woman’s ovum and allowed to develop and produce stem cells. The stem cells are removed from the pre-embryo and are treated to grown inyo whatever organ is needed. Thus, the new organ is transplanted into the patient. Stem cells

Possible Benifits -Cloned organs using therapeutic cloning is better than organs donated by another person: -No rejection of organ because the organ’s DNA would match the patient’s DNA -Patients do not need to wait for an organ donor to donate his/her organs -Brand new organs can work more efficiently than donated organs -Saving more lives, which otherwise would be lost due to the waiting for a transplant

Potential Problems -There are still many deficiencies/ethical disputes in therapeutic cloning: -Since therapeutic cloning is still in its early stages of development stem cells sometimes become mutated, thus rejected by the recipient’s body. -The production of stem cells needs to become more efficient. So far we need 100 eggs to produce one usable stem cell line. -Many pro-life supporters believe that human comes into existence at conception. When the stem cells are extracted the embryo dies, therefore pro-life supporters believe that this is murder.

Cloning (Somatic Cell Nuclear Transfer, SCNT)

Stem Cells Non-specialized cells that have the capacity to divide in culture and to differentiate into more mature cells with specialized functions Stem cells differ from other cells in three main ways. First, they are “unspecialized,” meaning they do not perform specialized functions, such as the way heart muscle cells help blood flow or red blood cells carry oxygen through the bloodstream. Second, under certain conditions, they can be transformed into cells with specialized functions. Third, these cells are capable of reproducing themselves over an extended period of time

Pluripotent cells Cells that are capable of self-renewal, and have broad potential to differentiate into multiple adult cell types. Pluripotent stem cells may be derived from somatic cell nuclear transfer or from surplus products of in vitro fertilization treatments when such products are donated under appropriate informed consent procedures.

Stem Cell Types 1. Adult Stem Cells 2. Embryonic Stem Cells – Derived through fertilization – Derived through nuclear transfer

Adult Stem Cell Undifferentiated cell found in a differentiated tissue in an adult organism that can renew itself and may, with certain limitations, differentiate to yield all of the specialized cell types of the tissue from which it originated

Adult Stem Cells Derived from adults Identified in some organs – Blood – Eye – Brain – Skeletal muscle – GI track (liver, pancreas, lumen) – Skin Significant limitations

Current Clinical Uses of Adult Stem Cells Cancers—Lymphomas, multiple myeloma, leukemias, breast cancer, neuroblastoma, renal cell carcinoma, ovarian cancer Autoimmune diseases—multiple sclerosis, systemic lupus, rheumatoid arthritis, scleroderma, scleromyxedema, Crohn’s disease Anemias (incl. sickle cell anemia) Immunodeficiencies—including human gene therapy Bone/cartilage deformities—children with osteogenesis imperfecta Corneal scarring-generation of new corneas to restore sight Stroke—neural cell implants in clinical trials Repairing cardiac tissue after heart attack—bone marrow or muscle stem cells from patient Parkinson’s—retinal stem cells, patient’s own neural stem cells, injected growth factors Growth of new blood vessels—e.g., preventing gangrene Gastrointestinal epithelia—regenerate damaged ulcerous tissue Skin—grafts grown from hair follicle stem cells, after plucking a few hairs from patient Wound healing—bone marrow stem cells stimulated skin healing Spinal cord injury—clinical trials currently in Portugal, Italy, S. Korea

Proposition 71: The California Stem Cell Research and Cures Act Acknowledges that stem cell research has the potential to produce new therapies States that “the federal government is not providing adequate funding…”; Prop 71 will “close the critical funding gap” Supports “stem cell research, emphasizing pluripotent stem cell and progenitor cell research and other vital medical technologies, for the development of live saving regenerative medical treatments and cures”