The development of the fertilized zygote into a complex organism has traditionally thought to be a unidirectional process, with cells in the developing fetus becoming gradually more committed to a specific tissue type. The recent development of mammalian cloning by nuclear transfer (NT) suggests that the mammalian oocyte has the remarkable ability to relieve the constraints imposed by cellular differentiation and return an adult nucleus to a totipotent, embryonic state. Thus, cloning by NT provides a unique opportunity to elucidate the molecular and cellular mechanisms by which an adult cell can be returned to an undifferentiated state, a process termed developmental reprogramming.
Aim 1) To determine whether mice can be cloned directly from terminally differentiated cells. Cloned mice have only been generated from terminally differentiated cells using an embryonic stem (ES) cell intermediate, suggesting that passage of the nucleus through an ES cell might be necessary for complete reprogramming. Experiments will be carried out to determine whether this is true or if instead, the oocyte and embryo alone can successfully reprogram the epigenetic state of a terminally differentiated nucleus.
Aim 2) To investigate if the transition from a differentiated state to a pluripotent state can be understood through the genome-wide changes in transcriptional activity taking place after NT. Towards this end, the genome wide changes in transcriptional activity taking place after NT are being assessed by microarray analysis. Experiments to characterize the functional importance of these observed gene expression changes between fertilized and cloned preimplantation embryos will be carried out. The experiments proposed here combine molecular, genetic and developmental approaches that will test the limits of reprogramming, elucidate mechanisms governing reprogramming and determine how inadequacies in reprogramming may lead to the inefficient nature of cloning. These studies may have profound importance for evaluating the medical utility of NT technology, deciphering the molecular basis of pluripotency and expanding our understanding of embryonic development and stem cell biology.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
1R01HD046732-01A1
Application #
6923279
Study Section
Special Emphasis Panel (ZRG1-DEV-1 (01))
Program Officer
Tasca, Richard J
Project Start
2005-04-01
Project End
2010-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
1
Fiscal Year
2005
Total Cost
$289,600
Indirect Cost
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Tabansky, Inna; Lenarcic, Alan; Draft, Ryan W et al. (2013) Developmental bias in cleavage-stage mouse blastomeres. Curr Biol 23:21-31
Navara, Christopher S; Hornecker, Jacey; Grow, Douglas et al. (2013) Derivation of induced pluripotent stem cells from the baboon: a nonhuman primate model for preclinical testing of stem cell therapies. Cell Reprogram 15:495-502
Mekhoubad, Shila; Bock, Christoph; de Boer, A Sophie et al. (2012) Erosion of dosage compensation impacts human iPSC disease modeling. Cell Stem Cell 10:595-609
Son, Esther Y; Ichida, Justin K; Wainger, Brian J et al. (2011) Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9:205-18
Boulting, Gabriella L; Kiskinis, Evangelos; Croft, Gist F et al. (2011) A functionally characterized test set of human induced pluripotent stem cells. Nat Biotechnol 29:279-86
Egli, Dieter; Chen, Alice E; Saphier, Genevieve et al. (2011) Reprogramming within hours following nuclear transfer into mouse but not human zygotes. Nat Commun 2:488
Egli, Dieter; Eggan, Kevin (2010) Recipient cell nuclear factors are required for reprogramming by nuclear transfer. Development 137:1953-63
Chen, Alice E; Egli, Dieter; Niakan, Kathy et al. (2009) Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines. Cell Stem Cell 4:103-6
Egli, Dieter; Sandler, Vladislav M; Shinohara, Mari L et al. (2009) Reprogramming after chromosome transfer into mouse blastomeres. Curr Biol 19:1403-9
Ichida, Justin K; Blanchard, Joel; Lam, Kelvin et al. (2009) A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog. Cell Stem Cell 5:491-503

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