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ABSTRACT: The production of transgenic mice using small DNA constructs has been widely used for many years to investigate the regulation
of gene activity. Small plasmid-based constructs (less than 20 kb) have been favored for a number of reasons, particularly
the ease with which they can be manipulated and purified in large quantities. While this approach is powerful, there are some
problems associated with the size of these transgenes. In particular, many of these small transgenes do not reproduce accurately
the expression seen from the endogenous gene. For some genes the regulatory elements that control activity are located at
a distance from the promoter and can be omitted from the transgene. These may be enhancers, repressors, boundary elements,
or even locus control regions (LCRs), which are responsible for maintaining the correct spatial and temporal expression patterns
of a number of genes, such as the globin clusters in mouse and humans (1). More important, small transgenes are susceptible
to position effects from the chromatin environment in which they integrate, which often results in either ectopic expression
(from trapping of nearby enhancers for other genes) or suppression of gene activity. Finally, small transgenes usually integrate
in a multicopy tandem arrangement that does not accurately reflect the situation seen at the endogenous locus. There is growing
evidence from studies in mouse and humans that the regulatory elements for many imprinted genes may be widely dispersed within
“imprinted domains,” which may span hundreds of kilobases (2,3). Therefore, it is unlikely that analysis of small transgenes
will provide much useful information concerning the expression or mechanism of imprinting for the majority of this unusual
class of genes.
02/2008: pages 55-65;
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ABSTRACT: MicroRNAs (miRNAs) have important roles in diverse cellular processes, but little is known about their identity and functions during early mammalian development. Here, we show the effects of the loss of maternal inheritance of miRNAs following specific deletion of Dicer from growing oocytes. The mutant mature oocytes were almost entirely depleted of all miRNAs, and they failed to progress through the first cell division, probably because of disorganized spindle formation. By comparing single-cell cDNA microarray profiles of control and mutant oocytes, our data are compatible with the notion that a large proportion of the maternal genes are directly or indirectly under the control of miRNAs, which demonstrates that the maternal miRNAs are essential for the earliest stages of mouse embryonic development.
Genes & Development 04/2007; 21(6):644-8. · 11.66 Impact Factor
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ABSTRACT: Cdkn1c encodes an embryonic cyclin-dependant kinase inhibitor that acts to negatively regulate cell proliferation and, in some tissues, to actively direct differentiation. This gene, which is an imprinted gene expressed only from the maternal allele, lies within a complex region on mouse distal chromosome 7, called the IC2 domain, which contains several other imprinted genes. Studies on mouse embryos suggest a key role for genomic imprinting in regulating embryonic growth and this has led to the proposal that imprinting evolved as a consequence of the mismatched contribution of parental resources in mammals.
In this study, we characterised the phenotype of mice carrying different copy number integrations of a bacterial artificial chromosome spanning Cdkn1c. Excess Cdkn1c resulted in embryonic growth retardation that was dosage-dependent and also responsive to the genetic background. Two-fold expression of Cdkn1c in a subset of tissues caused a 10-30% reduction in embryonic weight, embryonic lethality and was associated with a reduction in the expression of the potent, non-imprinted embryonic growth factor, Igf1. Conversely, loss of expression of Cdkn1c resulted in embryos that were 11% heavier with a two-fold increase in Igf1.
We have shown that embryonic growth in mice is exquisitely sensitive to the precise dosage of Cdkn1c. Cdkn1c is a maternally expressed gene and our findings support the prediction of the parental conflict hypothesis that that the paternal genome silences genes that have an inhibitory role in embryonic growth. Within the IC2 imprinted domain, Cdkn1c encodes the major regulator of embryonic growth and we propose that Cdkn1c was the focal point of the selective pressure for imprinting of this domain.
BMC Developmental Biology 02/2007; 7:53. · 2.79 Impact Factor
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ABSTRACT: The germ cell lineage is a specified cell population that passes through a series of differentiation steps before giving rise, eventually, to either eggs or sperm. We have investigated the manner in which primordial germ cells (PGCs) are reprogrammed in vitro to form pluripotent stem cells in response to exogenous fibroblast growth factor-2 (FGF-2). The response is dependent on time of exposure and concentration of FGF-2. PGCs isolated in culture show a motile phenotype and lose any expression of a characteristic germ cell marker, mouse vasa homolog. Subsequently, some but not all of the cells show further changes of phenotype, accompanied by changes in expression of endogenous FGF-2 and up-regulation of its receptor, fibroblast growth factor receptor-3, in the nucleus. We propose that it is from this reprogrammed component of the now heterogeneous PGC population that pluripotent stem cells arise.
Stem Cells 07/2006; 24(6):1441-9. · 7.78 Impact Factor
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ABSTRACT: The germ cell lineage is a specified cell population that passes through a series of differentiation steps before giving rise, eventually, to either eggs or sperm. We have investigated the manner in which primordial germ cells (PGCs) are reprogrammed in vitro to form pluripotent stem cells in response to exogenous fibroblast growth factor-2 (FGF-2). The response is dependent on time of exposure and concentration of FGF-2. PGCs isolated in culture show a motile phenotype and lose any expression of a characteristic germ cell marker, mouse vasa homolog. Subsequently, some but not all of the cells show further changes of phenotype, accompanied by changes in expression of endogenous FGF-2 and up-regulation of its receptor, fibroblast growth factor receptor-3, in the nucleus. We propose that it is from this reprogrammed component of the now heterogeneous PGC population that pluripotent stem cells arise.
Stem Cells 05/2006; 24(6):1441 - 1449. · 7.78 Impact Factor
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ABSTRACT: The relationship between germ cells and pluripotent embryonic stem (ES) cells is of particular interest, together with approaches to generate primordial germ cell (PGCs) from ES cells. A critical requirement in these experiments is the ability to unambiguously detect PGCs with the use of, for example, reporter genes. The currently available transgenic reporters do not show exclusive expression in PGCs at their earliest developmental stages. Here we describe the use of germline-restricted expression of stella, which is currently the best marker gene for PGCs. We generated two stella-GFP reporters and show that both transgenes surpass other reporters in terms of timing and specificity of expression in PGCs. Additionally, we demonstrate the usefulness of stella-GFP during the derivation of PGCs from ES cells.
genesis 03/2006; 44(2):75-83. · 2.53 Impact Factor
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ABSTRACT: Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers. This is followed by an initial PCR step to amplify the cDNAs and generate enough material to permit separate multiplex detection. The diluted initial PCR product is used as a template to check individual miRNA expression by real-time PCR. This sensitive technique permits miRNA expression profiling from a single cell, and allows analysis of a few cells from early embryos as well as individual cells (such as stem cells). It can also be used when only nanogram amounts of rare samples are available. The protocol can be completed in 7 d.
Nature Protocol 02/2006; 1(3):1154-9. · 8.36 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are a class of 17-25 nt non-coding RNAs that have been shown to have critical functions in a wide variety of biological processes during development. Recently developed miRNA microarray techniques have helped to accelerate research on miRNAs. However, in some instances there is only a limited amount of material available for analysis, which requires more sensitive techniques that can preferably work on single cells. Here we demonstrate that it is possible to analyse miRNA in single cells by using a real-time PCR-based 220-plex miRNA expression profiling method. Development of this technique will greatly facilitate miRNA-related research on cells, such as the founder population of primordial germ cells where rapid and dynamic changes occur in a few cells, and for analysing heterogeneous population of cells. In these and similar cases, our method of single cell analysis is critical for elucidating the diverse roles of miRNAs.
Nucleic Acids Research 02/2006; 34(2):e9. · 8.03 Impact Factor
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Yasuhide Ohinata,
Bernhard Payer,
Dónal O'Carroll,
Katia Ancelin,
Yukiko Ono,
Mitsue Sano, Sheila C Barton,
Tetyana Obukhanych,
Michel Nussenzweig,
Alexander Tarakhovsky,
Mitinori Saitou,
M Azim Surani
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ABSTRACT: Germ cell fate in mice is induced in pluripotent epiblast cells in response to signals from extraembryonic tissues. The specification of approximately 40 founder primordial germ cells and their segregation from somatic neighbours are important events in early development. We have proposed that a critical event during this specification includes repression of a somatic programme that is adopted by neighbouring cells. Here we show that Blimp1 (also known as Prdm1), a known transcriptional repressor, has a critical role in the foundation of the mouse germ cell lineage, as its disruption causes a block early in the process of primordial germ cell formation. Blimp1-deficient mutant embryos form a tight cluster of about 20 primordial germ cell-like cells, which fail to show the characteristic migration, proliferation and consistent repression of homeobox genes that normally accompany specification of primordial germ cells. Furthermore, our genetic lineage-tracing experiments indicate that the Blimp1-positive cells originating from the proximal posterior epiblast cells are indeed the lineage-restricted primordial germ cell precursors.
Nature 08/2005; 436(7048):207-13. · 36.28 Impact Factor
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ABSTRACT: Peg3 is an imprinted gene exclusively expressed from the paternal allele. It encodes a C(2)H(2) type zinc-finger protein and is involved in maternal behavior. It is important for TNF-NFkB signaling and p53-mediated apoptosis. To investigate the imprinting mechanism and gene expression of Peg3 and its neighboring gene(s), we used a 120 kb Peg3-containing BAC clone to generate transgenic mice. The BAC clone contains 20 kb of 5' and 80 kb of 3' flanking DNA, and we obtained three transgenic lines. In one of the lines harboring one copy of the transgene, Peg3 was imprinted properly. In the other two lines, Peg3 was expressed upon both maternal and paternal transmission. Imprinted expression was linked to the differential methylation of a region (DMR) upstream of the Peg3 gene. A second, maternally expressed gene, Zim1, present on the transgene was expressed irrespective of parental inheritance in all lines. These data suggest that, similar to other imprinted genes within domains, Peg3 and Zim1 are regulated by one or more elements lying at a distance from the genes. The imprinting of Peg3 seen in one line may reflect the presence of a responder sequence. Concerning the expression of the Peg3 transgene, we detected appropriate expression in the adult brain. However, this was not sufficient to rescue the maternal behavior phenotype seen in Peg3 deficient animals.
Mammalian Genome 05/2004; 15(4):284-95. · 2.89 Impact Factor
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ABSTRACT: stella is a novel gene specifically expressed in primordial germ cells, oocytes, preimplantation embryos, and pluripotent cells. It encodes a protein with a SAP-like domain and a splicing factor motif-like structure, suggesting possible roles in chromosomal organization or RNA processing. Here, we have investigated the effects of a targeted mutation of stella in mice. We show that while matings between heterozygous animals resulted in the birth of apparently normal stella null offspring, stella-deficient females displayed severely reduced fertility due to a lack of maternally inherited Stella-protein in their oocytes. Indeed, we demonstrate that embryos without Stella are compromised in preimplantation development and rarely reach the blastocyst stage. stella is thus one of few known mammalian maternal effect genes, as the phenotypic effect on embryonic development is mainly a consequence of the maternal stella mutant genotype. Furthermore, we show that STELLA that is expressed in human oocytes is also expressed in human pluripotent cells and in germ cell tumors. Interestingly, human chromosome 12p, which harbours STELLA, is consistently overrepresented in these tumors. These findings suggest a similar role for STELLA during early human development as in mice and a potential involvement in germ cell tumors.
Current Biology 01/2004; 13(23):2110-7. · 9.65 Impact Factor
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ABSTRACT: An early fundamental event during development is the segregation of germ cells from somatic cells. In many organisms, this is accomplished by the inheritance of preformed germ plasm, which apparently imposes transcriptional repression to prevent somatic cell fate. However, in mammals, pluripotent epiblast cells acquire germ cell fate in response to signalling molecules. We have used single cell analysis to study how epiblast cells acquire germ cell competence and undergo specification. Germ cell competent cells express Fragilis and initially progress towards a somatic mesodermal fate. However, a subset of these cells, the future primordial germ cells (PGCs), then shows rapid upregulation of Fragilis with concomitant transcriptional repression of a number of genes, including Hox and Smad genes. This repression may be a key event associated with germ cell specification. Furthermore, PGCs express Stella and other genes, such as Oct-4 that are associated with pluripotency. While these molecules are also detected in mature oocytes as maternally inherited factors, their early role is to regulate development and maintain pluripotency, and they do not serve the role of classical germline determinants.
Philosophical Transactions of The Royal Society B Biological Sciences 09/2003; 358(1436):1363-70. · 6.40 Impact Factor
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ABSTRACT: Germ cell fate in mice is induced in proximal epiblast cells by the extra-embryonic ectoderm, and is not acquired through the inheritance of any preformed germ plasm. To determine precisely how germ cells are specified, we performed a genetic screen between single nascent germ cells and their somatic neighbours that share common ancestry. Here we show that fragilis, an interferon-inducible transmembrane protein, marks the onset of germ cell competence, and we propose that through homotypic association, it demarcates germ cells from somatic neighbours. Using single-cell gene expression profiles, we also show that only those cells with the highest expression of fragilis subsequently express stella, a gene that we detected exclusively in lineage-restricted germ cells. The stella positive nascent germ cells exhibit repression of homeobox genes, which may explain their escape from a somatic cell fate and the retention of pluripotency.
Nature 08/2002; 418(6895):293-300. · 36.28 Impact Factor
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ABSTRACT: The reciprocally imprinted H19 and Igf2 genes form a co-ordinately regulated 130 kb unit in the mouse controlled by widely dispersed enhancers, epigenetically modified silencers and an imprinting control region (ICR). Comparative human and mouse genomic sequencing between H19 and Igf2 revealed two novel regions of strong homology upstream of the ICR termed H19 upstream conserved regions (HUCs). Mouse HUC1 and HUC2 act as potent enhancers capable of driving expression of an H19 reporter gene in a range of mesodermal tissues. Intriguingly, the HUC sequences are also transcribed bi-allelically in mouse and human, but their expression pattern in neural and endodermal tissues in day 13.5 embryos is distinct from their enhancer function. The location of the HUC mesodermal enhancers upstream of the ICR and H19, and their capacity for interaction with both H19 and Igf2 requires critical re-evaluation of the cis-regulation of imprinted gene expression of H19 and Igf2 in a range of mesodermal tissues. We propose that these novel sequences interact with the ICR at H19 and the epigenetically regulated silencer at differentially methylated region 1 (DMR1) of Igf2.
Development 04/2002; 129(5):1205-13. · 6.60 Impact Factor
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Andreas P. Russ,
Sigrid Wattler,
William H. Colledge,
Samuel A. J. R. Aparicio,
Mark B. L. Carlton,
Jonathan J. Pearce, Sheila C. Barton,
M. Azim Surani,
Kenneth Ryan,
Michael C. Nehls,
Valerie Wilson,
Martin J. Evans
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ABSTRACT: The earliest cell fate decision in the mammalian embryo separates the extra-embryonic
trophoblast lineage, which forms the fetal portion of the placenta, from the
embryonic cell lineages. The body plan of the embryo proper is established
only later at gastrulation, when the pluripotent epiblast gives rise to the
germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene
Nature 03/2000; 404(6773):95-99. · 36.28 Impact Factor
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CHRISTINE M. WILLIAMSON ,
NOBUAKI KIKYO ,
COLIN V. BEECHEY ,
SIMON T. BALL ,
ROSALIND M. JOHN , SHEILA C. BARTON ,
FUMITOSHI ISHINO ,
CHARLES TEASE ,
ELIZABETH R. MARTIN ,
BRUCE M. CATTANACH ,
M. AZIM SURANI ,
JOSEPHINE PETERS
Genetics Research 07/1997; 70(01):79 - 89. · 1.71 Impact Factor
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ABSTRACT: Complex phenotypes and genotypes characterize the human disease, Beckwith–Wiedemann syndrome (BWS). Genetic and epigenetic mutations are found in five different genes which all lie within a 1 Mb imprinted domain on human chromosome 11p15. Only two of these genes, p57 KIP2 ( CDKN1C ) and IGF2 , are likely to be functionally involved in this disease. The presence of the additional mutations therefore suggests a role for the regulation of these two genes by distant cis -elements. The mouse Igf2 gene is regulated by enhancers and imprinting elements which lie >120 kb downstream of its promoter. Here we show that key elements for expression of the mouse p57 Kip2 ( Cdkn1c ) gene also lie at a distance. Enhancers for expression within skeletal muscle and cartilage lie >25 kb downstream of the gene. In addition, we find no evidence for allele-specific expression of p57 Kip2 ( Cdkn1c ) from our bacterial artificial chromosome transgenes that span 315 kb around the locus. This suggests that a key imprinting element for p57 Kip2 ( Cdkn1c ) also lies at a distance. Therefore, BWS in humans may result from disruption of appropriate expression of the p57 KIP2 ( CDKN1C ) gene through mutations that occur at a substantial distance from the gene.
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ABSTRACT: Neuronatin (Nnat) is an imprinted gene that is expressed exclusively from the paternal allele while the maternal allele is silent and methylated. The Nnat locus exhibits some unique features compared with other imprinted domains. Unlike the majority of imprinted genes, which are organised in clusters and coordinately regulated, Nnat does not appear to be closely linked to other imprinted genes. Also unusually, Nnat is located within an 8-kb intron of the Bc10 gene, which generates a biallelically expressed, antisense transcript. A similar organisation is conserved at the human NNAT locus on chromosome 20. Nnat expression is first detected at E8.5 in rhombomeres 3 and 5, and subsequently, expression is widespread within postmitotic neuronal tissues. Using modified BAC transgenes, we show that imprinted expression of Nnat at ectopic sites requires, at most, an 80-kb region around the gene. Furthermore, reporter transgenes reveal distinct and dispersed cis-regulatory elements that direct tissue-specific expression and these are predominantly upstream of the region that confers allele-specific expression.
Developmental Biology.
