1. Instructor: Dr. Shahzad A. Mufti Advisor Department of Biosciences Cloning, Gene Therapy and Stem Cell Research

2. Genomic equivalence of all body cells. Spemann’s experiments of delayed nucleation. Amphibian cloning to determine whether or not nuclei undergo changes during development, Briggs & King(1950s) experiments on Rana. Nuclear isolation & transplantation into an enucleated & activated egg. Cloning, Gene Therapy and Stem Cell Research September 12, 20152

3. Cloning of mammals: Wilmut’s “Dolly” Nucleus from adult pregnant ewe mammary gland  into culture  at Gl, into enucleated egg (at second meiotic metaphase). Fusion of donor cells with enucleated egg through electric pulses, also egg activation. Developing embryos then transferred into uterus. One out of 434 eggs survived. Cloning, Gene Therapy and Stem Cell Research September 12, 20153

4. Mice, Cats, Cows also cloned. Many develop diseases/abnormalities. “Also not exactly identical” in all cases, due to random chromosomal events. Also faulty activation of genes, failure of histone modifications, methylation deficiencies. This cloning is NOT a good reproductive technology. Cloning, Gene Therapy and Stem Cell Research September 12, 20154

5. Embryonic Stem Cells (ES Cells) pluripotent, can be cultured for long period while remaining undifferentiated. ES cells obtained through 2 major techniques; from ICM of human embryos or from germ cells of aborted fetuses. Importance of ES cells in medicine. Can produce new population of cells to replace the deficient ones in sick people; such as Alzheimer, or Parkinson, or Spinal Cord Injury of Diabetics of Anemic etc. Embryonic Stem Cells and Therapeutic Cloning September 12, 20155

6. Quite successful in mice but not yet perfected in humans; histocompatibility still a problem, so rejection. Recent developments in “therapeutic cloning” patient specific-------so no rejection. Adult Stem Cells. Not successful yet. Difficult to isolate and grow. Also not “very pluripotent”. Partial success in Bone regeneration and Neural regeneration. Embryonic Stem Cells and Therapeutic Cloning September 12, 20156

7. Basic types of stem cells Totipotent stem cells Pluripotent stem cells Multipotent stem cells Unipotent stem cells 7

8. September 12, 20158

9. Hello Dolly !!!! Dolly the sheep – named after singer Dolly Parton - was created from a cell taken from a six-year- old donor sheep, using a technique called somatic cell nuclear transfer. The birth of the Finn Dorset sheep paved the way for other animals to be cloned.

10. Cumulina In 1997, scientists cloned the first mouse from adult cells, calling her Cumulina. Cumulina died at the age of two years and seven months – a relatively healthy age for a mouse. She gave birth to two litters during her lifetime. Mice remain one of the more difficult species to clone.

11. Endangered species In 2001, scientists announced the birth of a cloned gaur, a rare wild ox. Noah, was created from the skin cells of a male gaur that had died eight years previously; but Noah died just 48 hours after delivery. Cloning endangered species, or even species that are extinct, has been suggested as a method for conserving animal populations.

12. GM cows George and Charlie, born in 1998, were the first cloned transgenic calves, paving the way for "pharming". With "pharming", scientists hope to combine cloning and genetic modification to create animals capable of producing medicinal products. One of the calves' creators is now president of a company aiming to produce human antibodies.

13. When cloning goes wrong ….

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