1. Cell potential, animal cloning Can any cell develop into a complete organism? How do you clone a sheep?

2. Regenerating plants from a single cell

4. Testing the developmental potential of nuclei from differentiated cells (Needle prick can activate some eggs) (or suck out meiotic spindle with pipette) Does tadpole develop?

6. Copyright ©2003 by the National Academy of Sciences Gurdon, J. B. and Byrne, J. A. (2003) Proc. Natl. Acad. Sci. USA 100, 8048-8052 Fig. 2. (A) In Amphibia, nuclear transfer success declines rapidly as cells differentiate

7. Donor nucleusStages reached Zygoteblastula, tadpole, adult Blastulablastula, tadpole, adult Tadpole intestineblastula, tadpole, adult Cultured skin cellblastula (differentiated) Success of nuclear transplant experiments

8. Cleavage divisions in the frog Xenopus animal vegetal Blastula Why do frogs from nuclear transplant experiments often arrest at the blastula stage?

9. The mid-blastula transition (after about 12 rounds of cleavage) Growth phases added to Cell Cycle Embryo starts transcribing its own RNA

10. Donor nucleusStages reached Zygoteblastula, tadpole, adult Blastulablastula, tadpole, adult Tadpole intestineblastula, tadpole, adult Cultured skin cellblastula Blastula from aboveblastula, tadpole (see handout) Could some genes be missing from differentiated cells?

11. Conclusions of nuclear transplantation experiments in amphibians: Nuclei of differentiated cells have the potential to program development of many or all cells. (Therefore, the nuclei must contain all the necessary genes.) Their capacity to direct development is restricted as they differentiate. The ooctye cytoplasm can reprogram differentiated nuclei to allow them to direct complete development.

12. 07_37_Protein.produc.jpg Nuclear effect – ability of genes to be expressed Cytoplasm effects – signals to nucleus (transcription factors, signaling pathways)

13. 08_14_chromatin.struc.jpg Genes need to be accessible to be expressed

14. 05_30_histone tails.jpg Histone modification state may limit gene expression potential

15. DNA methylation state may limit gene expression potential

16. Cloning frogs from a single nuclear donor

17. 08_02_genetic.instruction.part2 Cloning mammals from adult cells (also activates egg)

18. (from Nature 405: 800-802)

20. NY Times, June 3, 2006 Clones' Debut Is a Test of Genetics, and Bettors' Wits By BILL FINLEY Anyone trying to select a winner at the mule races this weekend in Winnemucca, Nev., will no doubt have a hard time choosing between Idaho Gem and Idaho Star. They may have different names, but they are not necessarily different mules. Idaho Gem and Idaho Star are clones. They are two of three mules who were born in 2003 as the result of a cloning project at the University of Idaho and Utah State University. Leased from the University of Idaho for racing by Don Jacklin, an Idaho businessman, Gem and Star will make their first career starts today in separate 350-yard elimination races. The top eight finishers, based on time, will meet in tomorrow's final. There is parimutuel wagering on the races. Jacklin is confident the two mules will make the final, which could be part mule race, part science experiment. Who is faster, Idaho Gem or Idaho Star, or will they cross the finish line in unison? "Genetically, they should have equal ability," Jacklin said. "But you have to factor in the environmental effects. They can make a big difference." Jacklin counts his identical twin among the reasons that he is interested in the genetic sciences. He put up $400,000 to help create the clones. Dr. Gordon L. Woods, one of the veterinary scientists involved in the project, mated the parents of a champion mule named Taz and took cells from the resulting fetus. The cells were implanted in surrogates. Mules are a cross between female horses and male donkeys and are typically sterile.