1. DIFFERENTIATION

2. Differentiation The process by which cells or tissues of an organism acquire the ability to perform their special functions. The process by which cells or parts of an organism become different from one and other and also from their previous state.

3. How is this accomplished? Selective gene transcription In any given cell, only the genes necessary for basic metabolism and that cell’s special functions are active. muscle cells - actin, myosin pancreatic acinar cells - digestive enzymes neurons - tubulin, neurotransmitters

4. Mechanisms of selective gene transcription Ascaris suum, Parascalas univalens - nematode (roundworm) parasites found in pig intestine. http://cal.vet.upenn.edu/paraav/labs/lab_4.htm

5. *Chromosome diminution - loss of pieces of chromosomes during mitosis *In Ascaris, pieces of chromosomes are lost in certain cells during the 2nd - 5th cleavage divisions (the figure above which shows diminution during the 2nd cleavage is from the original work of Boveri (1925) and actually is for another roundworm, Parascalas univalens). *About 25% of the DNA is lost. This is mostly repetitive (non-gene sequence) DNA; however, a few functional genes are lost. *At the 32 cell stage only one cell remains with a complete genome. *This cell will give rise to the germ cell line. The other 31 cells give rise to the somatic tissues.

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7. Mechanisms of selective gene transcription Post-transcriptional control - mechanisms that act after the transcription event. e.g., Mechanisms that block the activity of specific mRNAs or proteins until a later time when they become active (e.g. maternally derived cytoplasmic information - formation of the primordial germ cells in amphibians and insects). Ultimate vs. Proximal controls of differentiation DNA transcription is the ultimate controlling factor in differentiation. However, the DNA is not always active in transcription at the time of the differentiation event. Proximal controls are the factors that are active at the time of the differentiation event. These could be genes or stored cytoplasmic mRNA that is activated at the time of differentiation.

8. Acetabularia crenulata http://www.biologe.de/Nuetzliches/botanik_online/d44/acetabul.htm Acetabularia mediterrania http://dragonja.nib.si/RtMadona/Slike_3.html http://imbi.uwc.ac.za/courses/ifaa/jpg/Berger/Berger_02.html Acetabularia

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12. Differentiation - some genes are turned off while others are allowed to function. Common dogma - most cells in the body contain a complete genome despite the fact that most of the genes in the genome are not necessary for each cells specific function. Is differentiation reversible? Is there some point in a cells differentiation where unnecessary genes become irreversibly inactive ( are irreversibly turned off)? Not too many years ago, the answer for mammalian cells would have been YES - however, things have changed.

13. Spemann, 1928

14. Gurdon, 1966 Somatic cell nuclear transfer.

15. Illmensee and Hoppe, 1981

16. Cell growth and division cycle

17. 1. Embryo splitting 1. Separate cells during early cleavage (2 cell - 4 cell) 2. Culture separate cells to blastocyst stage 3. Introduce cells in to uterus of hormonally prepared female 4. If successful, pregnancy and eventually birth 2. The Roslin Technique - used to create Dolly (Wilmut, 1997). 1. Collect oocyte from female 2. Enucleate oocyte 3. Induce adult cells in another culture to enter G o state 4. Fuse G o cell with enucleated oocyte with electrical shock Introduces adult cell nucleus into oocyte cytoplasm Electrical shock also activates egg -> Zygote 5. Cultures embyros to blastocyst stage 6. Introduce cells into uterus of hormonally prepared female 7. If successful, pregnancy and eventually birth (1 successful clone in 277 tries to get Dolly) Three cloning methods:

18. http://science.howstuffworks.com/cloning2.htm Wilmut et al., 1997 Cell induced to enter G phase. o The Roslin Technique

19. Hello Dolly - Dolly the sheep, the world's first clone from the nucleus of an adult mammal. http://www.guardian.co.uk/pictures/image/0,8543,-10904328739,00.html

20. 3. The Honolulu technique - Developed at University of Hawaii. This method was developed for cloning mice. 1. Use cells of the individual to be cloned that are naturally in the G o state (e.g. Sertoli cells, Neurons, Cumulus cells) to provide the nucleus. (somatic cell nuclear transfer) 2. Transfer the G o nucleus to an enucleated egg by injecting it into the egg. 3. Activate the egg using appropriate chemicals. 4. Culture to blastocyst stage and introduce into uterus of hormonally prepared female 5. If successful, pregnancy and eventually birth (3 successful clones for every 100 tries) Three cloning methods:

21. Mooving on - A pair of new-born cloned calves in a cowshed in Ishikawa Japan, on July 5 1998. Second adult mammal clones. Roslin Technique http://www.guardian.co.uk/pictures/image/0,8543,-10904328739,00.html

22. Idaho Gem The world's first cloned mule. The first clone to be born in the equine family. http://www.guardian.co.uk/pictures/image/0,8543,-10904328739,00.html

23. Copycat The world's first cloned kitten, named Cc. It was created by scientists in Texas using a cell taken from an adult tortoiseshell female. http://www.guardian.co.uk/pictures/image/0,8543,-10904328739,00.html

24. South Korea (Dr. Hwang Woo-Suk) unveils first dog clone Snuppy, whose name stands for Seoul National University puppy. Cloned using a cell taken from the ear of a three- year-old male Afghan hound. Scientists took nucleus from the ear cell and placed it into an enucleated egg cell. Honolulu Technique. This egg was then stimulated to start dividing and develop into an embryo. Once growing, it was transferred to Snuppy's surrogate mother, a yellow labrador. The Afghan pup was born by caesarean section after a full 60 days of pregnancy. Scandal followed - made false claims about producing human stem cell lines from cloned human embryos. http://news.bbc.co.uk/2/hi/science/nature/4742453.stm

25. Cloned goat Yangyang turns four Has given birth to twins and is now a great grandmother.

26. Five little piggies Five cloned female piglets, named Noel, Angel, Star, Joy and Mary. Scottish-based firm PPL Therapeutics says is a major step towards successfully producing animal organs and cells for use in human transplants. The pigs lack a gene to which the human immune system reacts aggressively. http://www.guardian.co.uk/pictures/image/0,8543,-10904328739,00.html

27. ANDi (inserted DNA spelled backwards) The first rhesus monkey cloned by embryo splitting. http://www.guardian.co.uk/pictures/image/0,8543,-10904328739,00.html In 2007, Shoukhrat Mitalipov at the Oregon Primate Research Center produced stem cells from cloned monkey embryos using the Honolulu technique.