1. Animal Cloning

2. Animal Clones Natural clones in animals are rarer than in plants, fungi and bacteria but they do occur. Thus – Hydra and Obelia will produce new organisms by budding – Aphids will produce large numbers of identical female offspring by parthenogenesis in the summer – Cleavage of a fertilised ovum to give two separate embryos will produce identical twins An animation on cloning can be viewed at: http://www.dnalc.org/resources/animations/cloning101.html http://www.dnalc.org/resources/animations/cloning101.html

3. Artificial cloning is becoming more commonplace and use one of two methods: – Artificial embryo splitting – Somatic cell nuclear transfer

4. Artificial Embryo Splitting This is the equivalent of artificially producing twins. – Cells of an early embryo are separated – Each cell can potentially grow into an individual – All will be genetically identical and a clone High value pedigree animals are used as egg and sperm donors – Produce a zygote which is grown onto the 8-, 16- or 32-cell stage – Embryo then split into single cells or groups of cells – Allowed to develop further before implantation into surrogate mothers – do not need to be high quality Cattle and sheep have been cloned in this way.

5. High quality cow  eggs Pedigree bull  sperm IVF Early embryo with 8, 16 or 32 cells Embryonic cells separated Embryos implanted into surrogate cows Cloned cattle produced

6. Q. Suggest which features might make the donor cow and the bull ‘high quality’. Q. Suggest why, following embryo splitting, the groups of cells are further grown on before implantation into the surrogates. Q. Why do the surrogates not have to be pedigree animals themselves to produce quality calves?

7. Q. Suggest which features might make the donor cow and the bull ‘high quality’. Milk yield; protein/fat content of milk; temperament; growth rate; food conversion rate; lean meat. Q. Suggest why, following embryo splitting, the groups of cells are further grown on before implantation into the surrogates. Check that growth and cell division are proceeding normally; may want to further subdivide embryos. Q. Why do the surrogates not have to be pedigree animals themselves to produce quality calves? Quality comes from the pedigree cow and bull; all embryos are genetically identical; surrogate simply acts as a womb for embryo to grow in.

8. Somatic Cell Nuclear Transfer In this technique: – A differentiated cell is extracted from an animal, and – Placed into an enucleated egg cell i.e. an egg cell that has had the nucleus removed With luck, the egg develops, but uses the instructions in the DNA from the donated nucleus to control its growth and development. The resulting animal is a clone of the animal from which the cell was removed from.

9. Somatic cell nuclear transfer produced Dolly the sheep in 1996, at the Roslin Institute, University of Edinburgh. – Dolly was the first mammal to be cloned from an adult cell. – Cells from the udder of a Finn Dorset ewe were fused with an enucleated egg taken from a Scottish Blackface ewe. Dolly was the only success from a large number of tries – 277 eggs were used to make 29 embryos which produced three lambs, only one of which, Dolly, survived. Dolly was fertile, went on to have lambs of her own, but got arthritis and was ‘put down’ aged six. Dolly also had a type of virus-induced lung cancer (sheep pulmonary adenomatosis) – Indoor-raised sheep are prone to it – Other sheep in the flock also suffered from it

10. Egg taken from a Scottish Blackface ewe Cell taken from udder of a Finn Dorset ewe Nucleus removed from egg Udder cell and enucleated egg fused together Embryo transplanted into uterus of Scottish Blackface ewe Finn Dorset lamb produced – a clone of the original, named ‘Dolly’ Electric shock and/or chemicals stimulate cell division

11. Q. Suggest why the surrogate mother was a Scottish Blackface rather than a Finn Dorset. Physical characteristics very different so it would be easy to spot lineage of the lamb; larger; known to be capable of accepting a transferred embryo. Q. Suggest how it might be possible to prove that Dolly was indeed a clone of the Finn Dorset sheep that donated the udder cells. Physical characteristics would confirm Finn Dorset ancestry; genetic fingerprinting; serotyping.

12. Non-Reproductive Clones Rather than produce a whole organism, the aim of non-reproductive cloning is to make cells, tissues and organs to replace those that have been damaged by disease, accident or age. Consequently, this is sometimes called therapeutic cloning. Various sources/options for the cloned cells: – Cancer cells – Repair faults with gene therapy – Stem cells

13. Cancer Cells Cancer cells are immortal – They can divide repeatedly and indefinitely. The most well known is the HeLa cell line – Taken from Henrietta Lacks in the early 1950s The cells have been used extensively in research and drugs testing.

15. Totipotentpluripotent Totipotent, pluripotent, uni/multipotent Anythingmany things Anything, many things, several things Pluripotent Multipotent / Unipotent Totipotent Potential for storage for future therapeutic use

16. Stem Cells Stem cells are capable of dividing and differentiating. There are: – Embryonic stem cells These are totipotent – Adult stem cells Usually either multi/unipotent or pluripotent However, there are exciting developments which may allow production of totipotent adult stem cells.

18. Windpipe transplant breakthrough It is flushed with chemicals to remove all existing cells Donor trachea "scaffold" coated with stem cells from the patient's hip bone marrow. Cells from the airway lining added Trachea is removed from dead donor patient Once cells have grown (after about four days) donor trachea is inserted into patient's bronchus

19. Trachea (wind-pipe) graft ready for transplantation Narrowing of bronchus to left lung http://news.bbc.co.uk/1/hi/health/7735696.stm

20. Potential Uses of Stem Cells Potential uses of stem cells are: – Regeneration of heart tissue damaged by heart attack – Production of liver to replace one damaged by disease – Repair nervous tissue damaged by multiple sclerosis – Repair of nervous tissue following a broken neck – Repair brain tissue in Parkinson’s disease There would be definite advantages to such uses – There would be no rejection – There would be no donor shortage – Stem cells are totipotent and so have great potential Synthetic organs seeded by stem cells: http://www.bbc.co.uk/news/health-14072829

22. Repair Faulty Genes in Gene Therapy Replace faulty gene in a cell line and then grow the repaired cells (nuclear reprogramming) Example: SCID (severe combined immunodeficiency disease) – Caused by faulty gene coding for an essential enzyme in T cells – Correct gene is inserted using a retrovirus – Repaired cells cloned before putting them back into the body

23. The stem cells should have the same recognition antigens as any other cell from the patient’s body

24. Advantages and Disadvantages of Animal Cloning Discuss the potential advantages and disadvantages of cloning animals, and then complete the summary table. You should consider: – Genetic uniformity – Desirable qualities – Ability to produce sufficient numbers – Specific examples of potential benefit

25. Advantages of Animal CloningDisadvantages of Animal Cloning Cloned animals are genetically identical – all have the good characteristics of their ‘parent’, e.g. milk yield, meat quality. Cloned animals are all genetically identical – may have unknown genetic disease or susceptibility to disease. Transgenic animals can be cloned, so producing a small herd that produces specific human protein in their milk, i.e. pharming. Quality of life of cloned animals may be poor – smaller groups, isolation, controlled conditions, shorter life expectancy.* Endangered animals can be cloned, so preserving their genes for future. Clones are genetically identical and so could be problems of genetic drift and evolutionary bottlenecks. Rapidly improve the quality of herds. Success rate is low for some types of cloning, e.g. Dolly only success out of 277 attempts. *Early evidence from cloning suggested lifespan may be less – thought that starts life with a genetic age that is the same as the cell that was cloned and that each cell/chromosome only capable of a certain number of divisions. May not be so!

26. Summary Depending upon your perspective a clone is: – A group of genetically identical organisms or – An exact copy of a: Cell Chromosome Gene DNA Reproductive cloning aims to produce a whole organism and can utilise natural or artificial methods. Non-reproductive cloning, aka therapeutic cloning, produces cells or tissues for treating disease. Natural plant clones are produced by vegetative propagation. – Examples include: Bulbs, corms tubers, runners, rhizomes and root suckers There are advantages: – Uniformity allows colonisation of an area that suited to – Resist damage by grazers – Fallback if sexual reproduction fails There are also disadvantages: – Uniformity means there is: Insufficient variation to cope with change Increased disease susceptibility – Takes time to colonise new areas

27. It is likely that all English elms are derived from trees introduced by the Romans – Only reproduces From root suckers Cuttings – All elms are a clone Susceptible to Dutch elm disease cause by the fungi – Ophiostoma ulmi and O. novo- ulmi and – Transmitted by the elm bark beetle Scolytus scolytus – English elm virtually wiped out by the disease Traditionally many cultivated varieties of plants have been propagated artificially – Cuttings Cut below a node and place in gritty compost Roots produced from cambium – Grafts Bud is grafted onto a root stock – Cuttings and grafts are identical to parent material Tissue culture is an example of artificial propagation – Meristem culture Buds are excised and grown into plantlets – Callus culture Explant material produces a mass of undifferentiated wound tissue (callus) Can be split and induced to differentiate

28. – Cell suspension Callus grown in suspension to obtain metabolites Natural clones in animals are rarer than in plants – Examples include: Identical twins Parthenogenesis in aphids Artificial cloning can be achieved by: – Artificial embryo splitting IVF using pedigree cow and bull Early embryo split into groups – 2, 4 or 8 cells – Each develops into further identical embryo – Somatic nuclear transfer Enucleated ovum has nucleus from mature cell inserted » Dolly the sheep is a well known example of such a clone » Low success rate for technique Non-reproductive cloning includes: – Use of cancer cells E.g. HeLa cells For testing drugs – Replacing faulty gene Then cloning repaired cell As with T cells of patients suffering from SCID – Stem cells To regenerate/repair tissue damaged by disease