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ABSTRACT: Small molecules will need to be identified and/or developed that target protein classes limiting reprogramming efficiency. A specific class of proteins includes epigenetic regulators that silence, or minimize expression, of pluripotency genes in differentiated cells. To better understand the role of specific epigenetic modulators in reprogramming, we have used shRNA delivered by lentivirus to assess the significance of individual epi-proteins in reprogramming pluripotent gene expression.
Methods in molecular biology (Clifton, N.J.) 01/2011; 702:299-316.
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ABSTRACT: Epigenetics refers to heritable changes in gene expression that are not attributable to changes in DNA sequence and impacts many areas of applied and basic biology including developmental biology, gene therapy, somatic cell nuclear transfer, somatic cell reprogramming, and stem cell biology. Epigenetic changes are known to contribute to aging in addition to multiple disease states. Epigenetic changes can be influenced by environmental factors that in turn can be inherited by daughter cells during cell division and can also be inherited through the germ line. Thus, it is intriguing to consider that epigenetics, in general, may play a role in human conditions that are strongly influenced by changes in the environment and lifestyle. In particular, metabolic syndrome, a condition increasing in prevalence around the world, is one such condition for which epigenetics is postulated to contribute. Epigenetic defects (epimutations) are thought to be more easily reversible (when compared with genetic defects) and, as such, have inspired efforts to identify novel compounds that correct epimutations or prevent progression to the disease state. These efforts have resulted in the development of a rapidly growing new field being referred to as epigenetic therapy. To date, 2 classes of drugs have received the most attention, that is, DNA methyltransferase inhibitors and histone deacetylase inhibitors; but recent data suggest that botanical sources may be a rich source of agents that can potentially modulate the epigenome and related pathways and potentially be useful in attenuating the progression of many factors related to development of metabolic syndrome. This review will provide an overview of the field of epigenetics, epigenetic therapy, and the molecules currently receiving the most interest with respect to treatment, and review data on botanical compounds that show promise in this regard.
Metabolism 07/2008; 57(7 Suppl 1):S16-23. · 2.66 Impact Factor
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ABSTRACT: While genetic studies have provided a wealth of information about health and disease, there is a growing awareness that individual characteristics are also determined by factors other than genetic sequences. These "epigenetic" changes broadly encompass the influence of the environment on gene regulation and expression and in a more narrow sense, describe the mechanisms controlling DNA methylation, histone modification and genetic imprinting. In this review, we focus on the epigenetic mechanisms that regulate adult (somatic) stem cell differentiation, beginning with the metabolic pathways and factors regulating chromatin structure and DNA methylation and the molecular biological tools that are currently available to study these processes. The role of these epigenetic mechanisms in manipulating adult stem cells is followed by a discussion of the challenges and opportunities facing this emerging field.
Critical Reviews in Eukaryotic Gene Expression 02/2008; 18(3):189-206. · 3.08 Impact Factor
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Jeffery M Betthauser,
Martha Pfister-Genskow,
Hongzhi Xu,
Paul J Golueke,
Jenine C Lacson,
Richard W Koppang,
Cena Myers,
Bing Liu,
Ina Hoeschele, Kenneth J Eilertsen,
Gregory H Leno
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ABSTRACT: Successful cloning by somatic cell nuclear transfer (NT) involves an oocyte-driven transition in gene expression from an inherited somatic pattern, to an embryonic form, during early development. This reprogramming of gene expression is thought to require the remodeling of somatic chromatin and as such, faulty and/or incomplete chromatin remodeling may contribute to the aberrant gene expression and abnormal development observed in NT embryos. We used a novel approach to supplement the oocyte with chromatin remodeling factors and determined the impact of these molecules on gene expression and development of bovine NT embryos. Nucleoplasmin (NPL) or polyglutamic acid (PGA) was injected into bovine oocytes at different concentrations, either before (pre-NT) or after (post-NT) NT. Pre-implantation embryos were then transferred to bovine recipients to assess in vivo development. Microinjection of remodeling factors resulted in apparent differences in the rate of blastocyst development and in pregnancy initiation rates in both NPL- and PGA-injected embryos, and these differences were dependent on factor concentration and/or the time of injection. Post-NT NPL-injected embryos that produced the highest rate of pregnancy also demonstrated differentially expressed genes relative to pre-NT NPL embryos and control NT embryos, both of which had lower pregnancy rates. Over 200 genes were upregulated following post-NT NPL injection. Several of these genes were previously shown to be downregulated in NT embryos when compared to bovine IVF embryos. These data suggest that addition of chromatin remodeling factors to the oocyte may improve development of NT embryos by facilitating reprogramming of the somatic nucleus.
Molecular Reproduction and Development 09/2006; 73(8):977-86. · 2.53 Impact Factor
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ABSTRACT: Common goals of microarray experiments are the detection of genes that are differentially expressed between several biological types and the construction of classifiers that predict biological type of samples. Here we consider a situation where there is no training data. There is considerable interest in comparing expression profiles associated with successful pregnancies (SP) and unsuccessful pregnancies (UP) in model and farm animals. Successful pregnancy rate is known to be much higher in embryos generated by in vitro fertilization (IVF) than in nuclear transfer (NT) embryos, and higher under induced ovulation for large follicles (LF) than for small follicles (SF). The tasks of identifying genes differentially expressed between SP and UP, and predicting SP for future samples are not well accomplished by comparing IVF and NT, or LF and SF. A suitable method is finite mixture model analysis (FMMA), which models each observed class (IVF and NT, or LF and SF) as a mixture of two distributions, one for SP and one for UP, with different known or unknown proportions (here known to be 0.50 SP for IVF and 0.02 SP for NT). The means of the two distributions differ for the differentially expressed genes, which we identify via a likelihood ratio test. We confirm by simulation that FMMA strongly outperforms hierarchical clustering and linear discriminant analysis using the known class labels (NT, IVF). We apply FMMA to a real data set on IVF and NT embryos, and compute their posterior probabilities of SP, which confirm our prior knowledge of the SP proportions for IVF and NT.
Veterinary Immunology and Immunopathology 06/2005; 105(3-4):187-96. · 2.08 Impact Factor
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Martha Pfister-Genskow,
Cena Myers,
Lynette A Childs,
Jenine C Lacson,
Thomas Patterson,
Jeffery M Betthauser,
Paul J Goueleke,
Richard W Koppang,
Gail Lange,
Patricia Fisher,
Steven R Watt,
Erik J Forsberg,
Ying Zheng,
Gregory H Leno,
Richard M Schultz,
Bing Liu,
Chiranjeet Chetia,
Xiao Yang,
Ina Hoeschele, Kenneth J Eilertsen
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ABSTRACT: Using an interwoven-loop experimental design in conjunction with highly conservative linear mixed model methodology using estimated variance components, 18 genes differentially expressed between nuclear transfer (NT)- and in vitro fertilization (IVF)-produced embryos were identified. The set is comprised of three intermediate-filament protein genes (cytokeratin 8, cytokeratin 19, and vimentin), three metabolic genes (phosphoribosyl pyrophosphate synthetase 1, mitochondrial acetoacetyl-coenzyme A thiolase, and alpha-glucosidase), two lysosomal-related genes (prosaposin and lysosomal-associated membrane protein 2), and a gene associated with stress responses (heat shock protein 27) along with major histocompatibility complex class I, nidogen 2, a putative transport protein, heterogeneous nuclear ribonuclear protein K, mitochondrial 16S rRNA, and ES1 (a zebrafish orthologue of unknown function). The three remaining genes are novel. To our knowledge, this is the first report comparing individual embryos produced by NT and IVF using cDNA microarray technology for any species, and it uses a rigorous experimental design that emphasizes statistical significance to identify differentially expressed genes between NT and IVF embryos in cattle.
Biology of Reproduction 04/2005; 72(3):546-55. · 4.01 Impact Factor
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ABSTRACT: To gain a better understanding of global methylation differences associated with development of nuclear transfer (NT)-generated cattle, we analyzed the genome-wide methylation status of spontaneously aborted cloned fetuses, cloned fetuses, and adult clones that were derived from transgenic and nontransgenic cumulus, genital ridge, and body cell lines. Cloned fetuses were recovered from ongoing normal pregnancies and were morphologically normal. Fetuses generated by artificial insemination (AI) were used as controls. In vitro fertilization (IVF) fetuses were compared with AI controls to assess effects of in vitro culture on the 5-methylcytosine content of fetal genomes. All of the fetuses were female. Skin biopsies were obtained from cloned and AI-generated adult cows. All of the adult clones were phenotypically normal and lactating and had no history of health or reproductive disorders. Genome-wide cytosine methylation levels were monitored by reverse-phase HPLC, and results indicated reduced levels of methylated cytosine in NT-generated fetuses. In contrast, no differences were observed between adult, lactating clones and similarly aged lactating cows produced by AI. These data imply that survivability of cloned cattle may be closely related to the global DNA methylation status. This is the first report to indicate that global methylation losses may contribute to the developmental failure of cloned bovine fetuses.
Biology of Reproduction 04/2003; 68(3):1009-14. · 4.01 Impact Factor
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Marvin M Pace,
Monica L Augenstein,
Jeffery M Betthauser,
Lynette A Childs, Kenneth J Eilertsen,
Joellyn M Enos,
Erik J Forsberg,
Paul J Golueke,
Doug F Graber,
John C Kemper, [......],
Gail Lange,
Tiffany L Lesmeister,
Kelly S Mallon,
Greg D Mell,
Pavla M Misica,
Martha Pfister-Genskow,
Nikolai S Strelchenko,
Gary R Voelker,
Steven R Watt,
Michael D Bishop
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ABSTRACT: Central to the success of large animal cloning is the production of healthy animals that can provide products for human health, food, and other animal agriculture applications. We report development of cloned cattle derived from 34 genetically unique, nonembryonic cell lines using nuclear transfer performed between 1 January 1998 and 29 February 2000. Nearly 25% (535/2170) of the recipients receiving reconstructed embryos initiated pregnancy. Overall, 19.8% (106/535) of the initiated pregnancies resulted in live births, while 77% (82/106) of these cattle clones remain healthy and productive today. Although a wide variation in birth weight of clone calves was observed, their growth rates, reproductive performance, and lactation characteristics are similar to that found in noncloned dairy cattle. Our data represent the most comprehensive information on cattle derived from nuclear transfer procedures and indicate that this emerging reproductive technology offers unique opportunities to meet critical needs in both human health care and agriculture.
Biology of Reproduction 08/2002; 67(1):334-9. · 4.01 Impact Factor
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Erik J Forsberg,
Nikolai S Strelchenko,
Monica L Augenstein,
Jeffery M Betthauser,
Lynette A Childs, Kenneth J Eilertsen,
Joellyn M Enos,
Todd M Forsythe,
Paul J Golueke,
Richard W Koppang,
Gail Lange,
Tiffany L Lesmeister,
Kelly S Mallon,
Greg D Mell,
Pavla M Misica,
Marvin M Pace,
Martha Pfister-Genskow,
Gary R Voelker,
Steven R Watt,
Michael D Bishop
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ABSTRACT: The pregnancy initiation and maintenance rates of nuclear transfer embryos produced from several bovine cell types were measured to determine which cell types produced healthy calves and had growth characteristics that would allow for genetic manipulation. Considerable variability between cell types from one animal and the same cell type from different animals was observed. In general, cultured fetal cells performed better with respect to pregnancy initiation and calving than adult cells with the exception of cumulous cells, which produced the highest overall pregnancy and calving rates. The cell type that combined relatively high pregnancy initiation and calving rates with growth characteristics that allowed for extended proliferation in culture were fetal genital ridge (GR) cells. Cultured GR cells used in nuclear transfer and embryo transfer initiated pregnancies in 40% of recipient heifers (197), and of all recipients that received nuclear transfer embryos, 9% produced live calves. Cultured GR cells doubled as many as 85 times overall and up to 75 times after dilution to single-cell culture. A comparison between transfected and nontransfected cells showed that transfected cells had lower pregnancy initiation (22% versus 32%) and calving (3.4% versus 8.9%) rates.
Biology of Reproduction 08/2002; 67(1):327-33. · 4.01 Impact Factor
