NuclearTransfer Wholeanimalcloning

What is a Clone?
Clone a group of two or more individuals with identical genetic (nuclear) makeup, derived by asexual reproduction, from a single common parent or ancestor.

Clones are NOT the exact copies

Asexual reproduction
Binary fission Bacteria and Archaea Budding Yeast, hydra Vegetative reproduction plants Spore formation fungi and some algae Fragmentation some animals (annelid worms and starfish), fungi, lichen and plants Parthenogenesis plants, invertebrates (e.g. water fleas, aphids, stick insects, some ants, bees and parasitic wasps), and vertebrates (e.g. some reptiles, amphibians, fish, very rarely birds)

Micropropagation
Micropropagation is the aseptic culture of cells, pieces of tissue, organs or whole plants. It is possible to regenerate new plants from small pieces of plant tissue. Why? Each cell of a plant:
has the same genetic makeup is totipotent has the ability to divide and produce all the differentiated cells in an organism, including extraembryonic tissues; it means the cell is capable of developing along a programmed pathway leading to the formation of an entire plant that is identical to the plant from which it was derived.

Totipotency of plant cells

meristems

and other tissues

Applications of micropropagation
Biotechnology Commercial propagation of plants:
a single plant can be used to propagate millions of new plants rapid multiplication speeds up the development of new cultivars can be used to develop and maintain virus-free stocks (meristem tissue is used, which typically is not-virusinfected)

Cultivated plants propagated by vegetative methods

Apple Pineapple Avocado Pear Banana Poplar Cacao Potato Canna Strawberry Cannabis Sugar cane Citrus (lemon, orange, grapefruit) Date Tea Fig Vanilla Grapes Willow Manioc (cassava) Nut crops (walnut, pecan) and many other species

WHOLEANIMALCLONING

Methods of animal cloning

Embryo splitting
to split an embryo into pieces, each forming a new embryo (usually, it is to split a 2 to 8 celled embryo before the cells differentiate). It is actually a simple form of cloning

Blastomere separation
outer coating is removed from 4-celled embryo, causing separation of individual embryo cells (bastomeres), which are then cultured in vitro to develop embryo clones.

Nuclear transfer
nucleus of an unfertilized egg is replaced with the nucleus of an donor cell.

Van Eenennaam, Clones 2008

using blastomers or cultured cells as nuclear donors

embryonic cells somatic cells

Nuclear transfer

ESC

Wolf et al., 1998

Somatic Cell Nuclear Transfer (SCNT or NTSC)

http://www.bootstrike.com/Genetics/StemCells/therapeutic_cloning.html

Chronology of advances in animal cloning

Hans Spemann (1938) proposed use of nuclear transfer to clone an organism.
This idea originates from theoretical considerations of an experimental method which would allow to determine whether a loss of genetic material occurs during cell differentiation. If occurs cloning would not be possible.

frog Rena pipiens (1952) Robert Briggs and Thomas King cloned northern leopard frogs
first animal cloning

frog Xenopus leavis (1958, 1962) John B. Gurdon

announced that he had cloned South African frogs using the nucleus of fully differentiated adult intestinal cells. This demonstrated that cells' genetic potential do not diminish as the cell became specialized.

carp (1963) controversial information sheep (1996) the first mammal cloned: Dolly mouse (1998) Wakayama, Perry and Yanagimachi
50 mice from adult cells (1997-1998)

Dolly
Dolly (July 5, 1996 February 14, 2003) - the first mammal to be cloned from an adult somatic cell, using the process of nuclear transfer. She was cloned by Ian Wilmut, Keith Campbell and colleagues at the Roslin Institute in Edinburgh, Scotland 277 oocytes were fused with cultured mammary gland cells: of these 29 reached morula/blastula stage, and were transferred into surrogate mothers leading to 13 pregnancies, but only one live birth.

During the Dolly`s lifetime, a lot of information on cloned organism development was collected. It was noticed that the age of the genetic material of cloned organisms is identical with the donor`s age. It means that biologically Dolly was 6 years old at her birth (age of the donor sheep). This causes that clone suffers from genetic disorders of mature/old-age donor and shows reduced disease resistance. At the age of five, Dolly developed arthritis. On February 14 , 2003 (at the age of 6 years and 7 months; the life-span for sheep: 11-12 years) Dolly was euthanized because of a progressive lung disease. She was bred with a Welsh Mountain ram and produced six lambs in total. Dolly was named after the country western singer Dolly Parton.

Chronology of advances in animal cloning

monkey Rhesus: from Tetra (female, 2000)
embryo splitting

pig (5 piglets Millie, Christa, Alexis, Carrel, and Dotcom from one sow; Scotland - 2000)

buffalo (male, 2001)

buffalo fetal fibroblasts used as donors

Chronology of advances in animal cloning

cow: Alpha and Beta (female, 2001)

cat: CopyCat CC a shorthaired calico (female, 2001)

mouse: in 2002 Hochedlinger and Jaenisch cloned mice from T lymphocytes: They showed that the genome of cloned individuals contains the same rearrangement of T lymphocyte receptor as the original population of lymphocytes. This finding is commonly considered to be the strongest prove for organism cloning from differentiated cells. rabbit: (2003) in France and South Korea

Chronology of advances in animal cloning

mule: Idaho Gem (male, May 2003) and Utah Pioneer (male, July 2003) deer: Dewey (2003) horse: Prometea an Arabian thoroughbred (female, 2003) rat: Ralph (male, 2003) fruit flies, Drosophila (2004) HUMAN (2004) Shin Yong Moon`s group They obtained pluripotent stem cells derived from cloned human blastocysts. dog: Snuppy Afghan hound (April 2005) wolf: grey wolves Snuwolf and Snuwolffy (2007), South Korea

Why develop cloning technology?

Potential applications of animal cloning

Rapidmultiplicationofdesiredlivestock Phenotypicevaluationandselection Animalconservation Transgenicapplications Humancellbasedtherapy

Potential applications of animal cloning

Rapid multiplication of desired livestock
rapid spread of superior genotypes production of large number of preferred sires for natural breeding (alternative to artificial insemination) rapid response to market changes

A 3-year-old Friesian dairy cow, and her genetically identical cloned calves.

Potential applications of animal cloning

Cloning for phenotypic evaluation and selection
to monitor effect of different environmental conditions on phenotypes of different lines of cloned animals
enhance genetic progress by increasing the accuracy of selection

Animal conservation
cryobanking of somatic cells from rare and endangered birds and animals insurance policy against further losses of diversity or possible extinction of wildlife to preserve endangered indigenous breeds of livestock adapted to particular environments
Lady, the first surviving cow of the Enderby Island breed (a rare breed of New Zealand origin, adapted to the harsh subantarctic environment), and Elsie her genetic duplicate.

Potential applications of animal cloning

Human cell-based therapy
to treat disorders in tissues that neither repair nor replace themselves:
diabetes, muscular dystrophy, spinal cord injury, certain cancers, various neurological disorders

healthy cells from patient could be used to produce therapeutic tissue for transplantation:
insulin-producing cells pancreatic cells for diabetes dopamine-producing nerve cells for Parkinson disease

this may also be associated with gene therapy to correct a genetic defect in the somatic cells (e.g. muscular dystrophy)

Potential applications of animal cloning

Transgenic applications:
cultured cells can be genetically modified in the lab the modifications can be checked prior to any animal work is conducted desired genetically modified cell lines may then be used in nuclear transfer nuclear transfer approach is more efficient than pronuclear injection method

Uses of animal cloning for transgenic applications

Pharmaceuticals
production of medically important molecules in milk, e.g.:
Human -1 antytrypsin for emphysema and cystic fibrosis treatment Human antithrombin III to regulate blood clotting during surgery

Nutraceuticals
improved milk products, e.g.
bovine milk proteins replaced with human equivalents different fatty acid profiles (CLA - conjugated linoleic acid isomers with anticancerogenic activity)

Agricultural production traits

genomics projects will identify target genes (influencing livestock production traits):
to improve feed conversion efficiency, growth rate, muscle composition confer disease and pest resitance

superior milk for cheese production

increased casein and reduced -lactoglobulin

Used of animal cloning for transgenic applications

Xenotransplantation
Shortages of human organs and tissues for transplantation Pigs are currently used for organ production
Availability Similar anatomy, organ size and physiology Problematic: disease (need to generate pathogen-free herds)

Modification of antigenes to reduce transplant rejection

Inactivation of -1,3-galactosyl transferase gene

Livestock as human genetic disease models

Sheep as alternative to mice models could be generated as superior research models:
Cystic fibrosis Huntington disease

Views on Cloning
In the debate over cloning there are those that feel that advances gained from cloning outweigh any social dilemmas. There are those that feel that cloning is wrong on a fundamental moral level and would produce scientific and social problems.

Pros on Cloning
Cloning will improve the overall quality of science and life. Cloning might produce greater understanding of the causes of miscarriages. Cloning experience may add to the increased understanding of genetics. Damage to nervous system could be treated through therapeutic cloning. Human cloning could be used for parents who risk passing a genetic defect to a child. Human cloning will allow a woman to have one set of identical twins.

Cons on Cloning
Those that do not agree with cloning feel that is an affront to religious societies. Cloning may reduce genetic variability. Cloning may cause people to settle for the best existing animals. Cloning is currently an expensive process. There is a risk of disease transfer. Any research into human cloning would eventually need to be tested on humans. Human cloning might be used to create a perfect human. Human cloning might have a detrimental effect on family relationships.

References
Campbell,1999,Nucleartransferinfarmanimalspecies.Sem.CellDevelop.Biol., 10:245252. Gurdon,1986,Nucleartransplantationineggsandoocytes,J.Cellscience,Suppl. 4:287318. Stephens,1995PlantmicropropagationusingAfricanVioletleaves www.biotech.iastate.edu/publications/lab_protocols/AV_Micropropagation.html VajtaandGjerris,2006,Scienceandtechnologyoffarmanimalcloning:Stateof theart,AnimalReproductionScience,92:211230. Wells,2002,Nucleartransferfromestablishedcelllines,EncyclopediaofLife Sciences,MacmillanPublisherGroup,NewYork,13:327331. Wilmut,2003,Wholeanimalcloning,EncyclopediaofLifeSciences,Macmillan PublisherGroup,NewYork Wolfetal.,1998,Nucleartransferinmammals:Recentdevelopmentsand perspectives,J.Biotechnology,65:99110. http://www.saskschools.ca/~stmarypa/bio30/animalcloning