Unit 1a Genomic Equivalence The Central Dogma Chromosome Puffs Gilbert 3e Fig 9 3 Bands genes Puffs active sites of transcription Conclusion Bands are the same puffs different This is evidence for differential gene expression 1950s Cloning by Nuclear Transfer Briggs King Step 1 enucleate activate the egg Gilbert 8e Fig 4 5 Cloning by Nuclear Transfer Briggs King Step 2 add donor nucleus Gilbert 8e Fig 4 5 Developmental Competence Decreases with the Age of the Nucleus Briggs King See Gilbert 8e Fig 4 6 Conclusion There must be changes in the nucleus that make it harder for nuclei from differentiated cells to direct full development But is it impossible 1960s Cloning by Nuclear Transfer Gurdon 2 nu a marker that can be assayed under the microscope Gilbert 6e Fig 4 9 Cloning by Nuclear Transfer Gurdon Conclusion rarely a nucleus from an adult cell can direct normal development Gilbert 6e Fig 4 9 Cloning by Nuclear Transfer Gurdon Gilbert 8e Fig 4 7 Conclusion rarely a nucleus from an adult cell can direct normal development Cloning by Nuclear Transfer S Willadsen and others Wilmut et al 2002 Nature 419 583 586 Conclusion Developmental competence of donor nucleus decreases with age of donor cell in mammals too 1997 Cloning Dolly 1997 Cloning Dolly Dr Ian Wilmut Dolly Conclusion rarely a nucleus from an adult mammalian cell can direct normal development Dolly and egg surrogate Cloning Dolly Gilbert 10e Fig 2 1 Cloning Dolly Gilbert 10e Fig 2 1 Dolly Are Clones Healthy Dolly was fertile Dolly Bonnie Possible cloning related abnormalities 10 shorter telomeres arthritis eventually euthanized http www scq ubc ca targetingtelomeres and cancer for dummi s 1998 Cloning Possible in Mice Gilbert 6e Fig 4 10 Cloning Possible in Mice Gilbert 6e Fig 4 10 Cloning Possible in Mice Cumulina Cumulina Surrogate Dr T Yanagimachi Offspring 1997 98 Beefing Up Cloning becomes possible in many livestock species Fibroblasts non quiescent Resulting calves Conclusion Telomere length appeared normal therefore Dolly may be an exception Beefing Up Cloning Technology Old fibroblasts can result in seemingly healthy calves Clone of a clone Beefing Up Cloning Technology 2008 Cloning of Pets Becomes Possible Cats CC 2002 The Clone kitten CC Gilbert 9e Fig 2 2 The donor Rainbow Cloning Dogs January 2009 The 155 000 dog Lancelot Encore Attempts to Use Cloning for Species Conservation 2001 2003 Noah a cloned gaur 2001 Cloned banteng 2003 Conclusion Many attempts at reproductive cloning fail Cloning for Conservation 2013 Pyrenean Ibex http www bbc com news science environment 25052233 Cloning Technology Clones from the Frozen Mouse frozen for 16 years Clone Wakayama et al 2008 PNAS 105 17318 22 Exotic Uses of Cloning Technology Exotic Uses of Cloning Technology Ice Baby Ice baby http ngm nationalgeographic com 2009 05 mammoths mueller text Why Is Cloning So Rarely Successful Large Offspring Syndrome LOS https formation ens lyon fr Formation Groupes Formation Continue Sciences de la vie Determinisme du sexe et clonage ClonageTransgenese ProblemesClones htm Why Is Cloning So Rarely Successful Placental Defects in Clones clone normal Hill et al 2000 Biol Reprod 63 1787 1794 Conclusion Developmental defects often arise during organ formation including formation of the placenta a structure derived from the embryo s cells Why Do Clones Die Defects in chemical status of chromatin DNA and associated proteins called histones Chemical status of DNA is established separately from simple transfer of chromosomes to offspring On top of genetics epigenetics We ll learn more about this in our molecular genetics lectures Epigenetic Status of Chromatin Normal Jaenisch R 2004 New Engl J Med 351 2787c Chemical modification of DNA bases methylation and associated histone proteins methylation acetylation occurs during normal early development The chemical status of DNA affects its ability to be transcribed into RNA Epigenetic Status of Chromatin Normal Embryos These changes in DNA occur over many rounds of cell division and hence many months Chromatin Status Nuclear Transfer Embryos In SCNT a nucleus with an established pattern of methylation of its DNA is placed into a donor oocyte without opportunity for normal processes that establish methylation patterns to operate Chromatin Status Nuclear Transfer Embryos It is therefore much harder to appropriately reset the methylation status of DNA in the cloned embryo compared with a normal embryo recall Dolly was 1 277 attempts Epigenetic Defects Lead to Embryo Failure Rhind et al 2003 Nature Rev Gen 4 855 864 Why Clone Humans Why Clone Therapeutic vs Reproductive Reproductive Adult cell 2n Nuclear transfer Therapeutic Patient s cell 2n Enculeated oocyte Nuclear transfer Nuclear transfer embryo 2n Nuclear transfer embryo 2n Nucleartransfer blastocyst Nucleartransfer blastocyst Enculeated oocyte ES cells from blastocyst Transfer into uterus of surrogate mother Adapted from Jaenisch R 2004 New Engl J Med 351 2787 Therapeutic Cloning The goal genetically matched cells to replace or repair damaged cells Jaenisch R 2004 NEJM 351 2787 June 1997 Assessment of Human Cloning National Bioethics Advisory Commission Harold Shapiro Alta Charo A t this time it is morally unacceptable for anyone in the public or private sector whether in a research or clinical setting to attempt to create a child using somatic cell nuclear transfer cloning Couldn t agree on ban beyond 5 yr moratorium 1998 Inter Species Cloning Advanced Cell Technologies July 2001 Assessments of Human Cloning Bush Administration said it unequivocally is opposed to the cloning of human beings either for reproduction or for research The moral and ethical issues posed by human cloning are profound and cannot be ignored in the quest for scientific discovery U S House passed ban on cloning of all kinds Note U S Senate passed no such ban therefore cloning is not illegal in the U S Nov 2001 ACT Cloning in Humans Advanced Cell Technologies ACT reports first attempt at therapeutic cloning Nov 2001 ACT Cloning in Humans President Bush s Response We should not as a society grow life to destroy it And that s exactly what s taking place And I have made that position very clear I haven t changed my mind And this evidence today that they re trying to achieve that objective to grow an embryo in order to extract a stem cell in order for that embryo to die is bad public policy Not only that it s morally wrong in my opinion Jan 18 2002 Assessments of Human Cloning