The aim of this project is to generate important new insights concerning epigenetic factors in oocytes governing reprogramming following somatic cell nuclear transfer (SCNT) and formation and maintenance of the first two lineages in mammalian embryos, the inner cell mass (ICM) and trophectoderm (TE). Our main hypothesis is that experimental modulation of expression levels for the key transcription factors in oocytes will prevent formation of the TE while enhancing the production of the ICM lineage. These alterations should preclude the formation of an embryo while promoting and enhancing donor nucleus reprogramming required for the formation of a single lineage from which pluripotent stem cell lines can be established. We propose the following specific aims:
Aim 1. Establish a mouse model of ANT with improved ESC derivation. Initially, we will investigate the effect of siRNA-mediated knockdown of Cdx2, Tead4, and E-Cadherin in mouse oocytes on reprogramming of somatic cells after SCNT. Next, we will examine a cumulative impact of the TE inactivation along with upregulation of critical pluripotent factors - Oct4, Sox2, and Nanog - on reprogramming and derivation of ESCs.
Aim 2. Define factors in monkey oocytes that are crucial for TE development. In Experiment 1, we will examine the presence and expression profiles of TEAD4 in monkey oocytes and preimplantation embryos. Next, we will study the role of TEAD4 in monkey oocytes and embryos by morpholino-assisted knockdown. Lastly, fertilized TEAD4-deficient oocytes will be used for ESC isolation.
Aim 3 : Analyze the roles of key maternal TE factors in epigenetic reprogramming after monkey SCNT. In this aim, we will apply most promising approaches to assess the validity of ANT in the monkey model by first creating CDX2 and TEAD4 depleted oocytes and then conducting SCNT and subsequently deriving ESCs from the resultant pluripotent cells.

Public Health Relevance

In this application we will study the potential of autologous (patient-matched) stem cells derived by somatic cell nuclear transfer in the nonhuman primate model while avoiding the destruction of viable embryos. The outcomes of these experiments, in turn, will allow the production of useful preclinical monkey models for testing therapeutic applications involving autologous stem cells for the treatment of wide range of degenerative diseases.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD059946-04
Application #
8431435
Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
Program Officer
Ravindranath, Neelakanta
Project Start
2010-02-01
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2013
Total Cost
$529,101
Indirect Cost
$157,350
Name
Oregon Health and Science University
Department
Obstetrics & Gynecology
Type
Other Domestic Higher Education
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Mitalipov, Shoukhrat; Wolf, Don P (2014) Clinical and ethical implications of mitochondrial gene transfer. Trends Endocrinol Metab 25:5-7
Mitalipov, Shoukhrat; Amato, Paula; Parry, Samuel et al. (2014) Limitations of preimplantation genetic diagnosis for mitochondrial DNA diseases. Cell Rep 7:935-7
Daughtry, Brittany; Mitalipov, Shoukhrat (2014) Concise review: parthenote stem cells for regenerative medicine: genetic, epigenetic, and developmental features. Stem Cells Transl Med 3:290-8
Wolf, Don P; Mitalipov, Shoukhrat (2014) Mitochondrial replacement therapies can circumvent mtDNA-based disease transmission. Cell Metab 20:6-8
Kang, Eunju; Wu, Guangming; Ma, Hong et al. (2014) Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos. Nature 509:101-4
Polejaeva, Irina; Mitalipov, Shoukhrat (2013) Stem cell potency and the ability to contribute to chimeric organisms. Reproduction 145:R81-8
Wu, Guangming; Han, Dong; Gong, Yu et al. (2013) Establishment of totipotency does not depend on Oct4A. Nat Cell Biol 15:1089-97
Tachibana, Masahito; Amato, Paula; Sparman, Michelle et al. (2013) Towards germline gene therapy of inherited mitochondrial diseases. Nature 493:627-31
Tachibana, Masahito; Amato, Paula; Sparman, Michelle et al. (2013) Human embryonic stem cells derived by somatic cell nuclear transfer. Cell 153:1228-38
Tachibana, Masahito; Sparman, Michelle; Ramsey, Cathy et al. (2012) Generation of chimeric rhesus monkeys. Cell 148:285-95

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