This document contains abstracts from two studies:
1. The first study identifies a hypoxia/HIF/Kdm3a pathway that controls trophoblast stem cell differentiation and organization of the hemochorial placenta. This pathway regulates genes involved in trophoblast invasion and vascular remodeling during pregnancy.
2. The second study examines endometrial gene expression in lactating cows, dry cows, and heifers on day 19 of pregnancy. The study finds 135 differentially expressed genes between lactating cows and heifers, but only 17 between dry cows and heifers, suggesting lactation impacts the endometrial response to the developing embryo. Functional analysis found effects on vesicle transport and cytokine signaling
CELLULAR REPROGRAMMING: Current Technology, Perspectives and Generation of iP...Munna Yadav
Reprogramming refers to erasure and remodelling of epigenetic marks, such as DNA methylation, during mammalian development. Exposure of a differentiated cell nucleus to the cytoplasm of less differentiated cell leads to erasure of the stable epigenetic code that maintains the differentiated cell’s phenotype. Gradually, the nucleus acquires a new epigenetic code that is characteristic of the dedifferentiated cell donating the cytoplasm, a process termed cellular reprogramming.
Introduction
Genetics of somatic cell
Somatic cell genetics
Somatic cell nuclear transfer
Somatic cell hybridization
Mapping human genes by using human rodent hybrids
In medical application
Production of monoclonal antibodies by using hybridoma technology
Conclusion
References
Variant G6PD levels promote tumor cell proliferation or apoptosis via the STA...Enrique Moreno Gonzalez
Glucose-6-phosphate dehydrogenase (G6PD), elevated in tumor cells, catalyzes the first reaction in the pentose-phosphate pathway. The regulation mechanism of G6PD and pathological change in human melanoma growth remains unknown.
CELLULAR REPROGRAMMING: Current Technology, Perspectives and Generation of iP...Munna Yadav
Reprogramming refers to erasure and remodelling of epigenetic marks, such as DNA methylation, during mammalian development. Exposure of a differentiated cell nucleus to the cytoplasm of less differentiated cell leads to erasure of the stable epigenetic code that maintains the differentiated cell’s phenotype. Gradually, the nucleus acquires a new epigenetic code that is characteristic of the dedifferentiated cell donating the cytoplasm, a process termed cellular reprogramming.
Introduction
Genetics of somatic cell
Somatic cell genetics
Somatic cell nuclear transfer
Somatic cell hybridization
Mapping human genes by using human rodent hybrids
In medical application
Production of monoclonal antibodies by using hybridoma technology
Conclusion
References
Variant G6PD levels promote tumor cell proliferation or apoptosis via the STA...Enrique Moreno Gonzalez
Glucose-6-phosphate dehydrogenase (G6PD), elevated in tumor cells, catalyzes the first reaction in the pentose-phosphate pathway. The regulation mechanism of G6PD and pathological change in human melanoma growth remains unknown.
Induced Pluripotent Stem-Like Cells Derived from Ban, a Vietnamese Native Pig...AI Publications
Induced pluripotent stem cells (iPSc) is a promising technology for applying in bio-medicine and biodiversity conservation. In the present study, we isolate and culture fibroblasts from Ban – a Vietnamese native pig breed and transfer episomal plasmid containing genes Oct3/4, Sox2, Klf4, l-Myc, LIN28 and EBNA1 in order to reprogram cells. We isolated, cultured and cryopreserved successfully 9 primary fibroblast lines from Ban (culture percentage is 90.0%). Plasmids was successfully transferred into Ban fibroblasts with high efficiency. Changes in morphology of fibroblasts into pluripotent stem-like cells showed that they had been reprogrammed under the effect of transferred genes. The pluripotency signal was further proved by in vitro differentiation by formation of embryoid body in all 3 transfected cell lines. The results showed that pluripotent stem-like cells has successfully derived in Ban pigs.
Understanding of Models use for biomedical research who have similar physiological function like humans ,and the how to generate and which models are useful
Annals of Mutagenesis is an open access, peer reviewed, scholarly journal dedicated to publish articles covering all areas of Mutagenesis.
The journal aims to promote research communications and provide a forum for doctors, researchers, physicians and healthcare professionals to find most recent advances in all areas of Mutagenesis. Annals of Mutagenesis accepts original research articles, reviews, mini reviews, case reports and rapid communication covering all aspects of mutagenesis.
Annals of Mutagenesis strongly supports the scientific up gradation and fortification in related scientific research community by enhancing access to peer reviewed scientific literary works. Austin Publishing Group brings universally peer reviewed journals under one roof thereby promoting knowledge sharing, mutual promotion of multidisciplinary science.
Induced Pluripotent Stem-Like Cells Derived from Ban, a Vietnamese Native Pig...AI Publications
Induced pluripotent stem cells (iPSc) is a promising technology for applying in bio-medicine and biodiversity conservation. In the present study, we isolate and culture fibroblasts from Ban – a Vietnamese native pig breed and transfer episomal plasmid containing genes Oct3/4, Sox2, Klf4, l-Myc, LIN28 and EBNA1 in order to reprogram cells. We isolated, cultured and cryopreserved successfully 9 primary fibroblast lines from Ban (culture percentage is 90.0%). Plasmids was successfully transferred into Ban fibroblasts with high efficiency. Changes in morphology of fibroblasts into pluripotent stem-like cells showed that they had been reprogrammed under the effect of transferred genes. The pluripotency signal was further proved by in vitro differentiation by formation of embryoid body in all 3 transfected cell lines. The results showed that pluripotent stem-like cells has successfully derived in Ban pigs.
Understanding of Models use for biomedical research who have similar physiological function like humans ,and the how to generate and which models are useful
Annals of Mutagenesis is an open access, peer reviewed, scholarly journal dedicated to publish articles covering all areas of Mutagenesis.
The journal aims to promote research communications and provide a forum for doctors, researchers, physicians and healthcare professionals to find most recent advances in all areas of Mutagenesis. Annals of Mutagenesis accepts original research articles, reviews, mini reviews, case reports and rapid communication covering all aspects of mutagenesis.
Annals of Mutagenesis strongly supports the scientific up gradation and fortification in related scientific research community by enhancing access to peer reviewed scientific literary works. Austin Publishing Group brings universally peer reviewed journals under one roof thereby promoting knowledge sharing, mutual promotion of multidisciplinary science.
Female mammals achieve dosage compensation by inactivating one of their two X chromosomes
during development, a process entirely dependent on Xist, an X-linked long noncoding
RNA (lncRNA). At the onset of X chromosome inactivation (XCI), Xist is up-regulated
and spreads along the future inactive X chromosome. Contextually, it recruits repressive
histone and DNA modifiers that transcriptionally silence the X chromosome. Xist regulation is
tightly coupled to differentiation and its expression is under the control of both pluripotency
and epigenetic factors. Recent evidence has suggested that chromatin remodelers accumulate
at the X Inactivation Center (XIC) and here we demonstrate a new role for Chd8 in Xist
regulation in differentiating ES cells, linked to its control and prevention of spurious
transcription factor interactions occurring within Xist regulatory regions. Our findings have a
broader relevance, in the context of complex, developmentally-regulated gene expression.
cloning. Second, it is sensitive. Activities canbe detected WilheminaRossi174
cloning. Second, it is sensitive. Activities can
be detected in the purified GST-ORF pools
that simply cannot be detected in extracts or
cells, the starting point of both conventional
purification and expression cloning. Because
the GST-ORFs are individually expressed at
high levels and are largely free of extract
proteins after purification, activities can be
measured for hours without competing activ-
ities that destroy the substrate, the product, or
the enzymes.
In addition to the conventional use demon-
strated here, this array could be used in two
other ways: (i) to determine the range of poten-
tial substrate proteins for any protein-modifying
enzyme (such as a protein kinase) before genet-
ic or biochemical tests to establish authentic
substrates and (ii) to identify genes encoding
proteins that bind any particular macromole-
cule, ligand, or drug. Thus, one could rapidly
ascribe function to many presently unclassified
yeast proteins, complementing other genomic
approaches to deduce gene function from ex-
pression patterns, mutant phenotypes, localiza-
tion of gene products, and identification of in-
teracting partners.
References and Notes
1. H. Simonsen and H. F. Lodish, Trends Pharmacol. Sci.
15, 437 (1994).
2. Plasmid pYEX 4T-1 (Clontech, Palo Alto, CA) was
modified by the addition of a 140-nucleotide recom-
bination domain, 39 of its Eco RI site, linearized within
the recombination domain by restriction digestion,
and cotransformed with a genomic set of reamplified
ORFs that had the same ends as the linearized plas-
mid [ J. R. Hudson Jr. et al., Genome Res. 7, 1169
(1997)] into strain EJ 758 [MATa his3-D200, leu2-
3,112, ura3-52, pep4::URA3], a derivative of JHRY-
20-2Ca (5). Transformants obtained on synthetic
minimal (SD) 2 Ura drop-out plates [F. Sherman,
Methods Enzymol. 194, 3 (1991)] (.100 in all cases,
and more than five times the cut vector in 97% of the
cases) were eluted in batch and saved in 96-well
microtiter plates. The library contains 6080 ORF-
containing strains and 64 strains with vector only.
3. Cell patches were inoculated in SD 2 Ura liquid
medium, grown overnight, reinoculated, and grown
overnight in SD 2 Ura 2 Leu medium, and then
inoculated into 250 ml of SD 2 Ura 2 Leu medium,
grown to absorbance at 600 nm of 0.8, and induced
with 0.5 mM copper sulfate for 2 hours before har-
vest [I. G. Macreadie, O. Horaitis, A. J. Verkuylen,
K. W. Savin, Gene 104, 107 (1991)]. Cells were re-
suspended in 1 ml of buffer [50 mM tris-HCl (pH 7.5),
1 mM EDTA, 4 mM MgCl2, 5 mM dithiothreitol (DT T),
10% glycerol, and 1 M NaCl] containing leupeptin (2
mg/ml) and pepstatin (1 mg/ml), and extracts were
made with glass beads [S. M. McCraith and E. M.
Phizicky, Mol. Cell. Biol. 10, 1049 (1990)], followed
by supplementation with 1 mM phenylmethylsulfo-
nyl fluoride and centrifugation. GST-ORF fusion pro-
teins were purified by glutathione agarose chroma-
tography in buffer containing 0.5 M NaCl, essentially
as described [ J. ...
Similar to SSR 2015-poster-A Hypoxia-HIF-Kdm3a Pathway Controls Trophoblast Stem Cell Lineage Decisions and Organization of the Hemochorial Placenta (20)
SSR 2015-poster-A Hypoxia-HIF-Kdm3a Pathway Controls Trophoblast Stem Cell Lineage Decisions and Organization of the Hemochorial Placenta
1.
2015 Abstracts – Page 44
115. A Hypoxia/HIF/Kdm3a Pathway Controls Trophoblast Stem Cell Lineage Decisions and Organization of the Hemochorial
Placenta.
Damayanti Chakraborty1
, Wei Cui2
, Regan Scott1
, Pramod Dhakal1
, Stephen Renaud1
, Gracy Rosario3
, Jay Vivian4
, Makoto Tachibana5
,
Mohammad K. Rumi4
, Michael Soares6
.
1
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA; 2
University of
Massachusetts, Amherst, Boston, MA, USA; 3
NA, Mumbai, Maharashtra, India; 4
Pathology and Laboratory Medicine, University of
Kansas Medical Center, Kansas City, KS, USA; 5
Institute for Enzyme Research,The University of Tokushima, Tokushima, Tokushima,
Japan; 6
IRHRM, University of Kansas Medical Center, Kansas City, KS, USA
The hemochorial placenta develops from the coordinated multi-lineage differentiation of trophoblast cells. Specific trophoblast
cell lineages are organized within the placentation site to perform specialized functions. The invasive trophoblast lineage remodels uterine
spiral arteries to convert them to flaccid low resistance vessels, facilitating the flow of nutrients to the placenta and fetus. Failure of
trophoblast invasion and vascular remodeling is associated with pathological conditions such as preeclampsia, intrauterine growth
restriction, and preterm birth. Oxygen delivery represents an environmental stimulus with an instructive role influencing trophoblast cell
differentiation and organization of the hemochorial placentation. In this study, we perform a series of in vitro and in vivo experiments
delineating a hypoxia- activated regulatory pathway controlling hemochorial placentation. Key downstream events are delineated using rat
trophoblast stem (TS) cells and tested in vivo using trophoblast-specific lentiviral gene delivery and genome editing. Initially, DNA
microarray analyses were performed on rat TS cells exposed to ambient or low oxygen (0.5%). Upregulation of genes characteristic of an
invasive/vascular remodeling/inflammatory phenotype and a marked downregulation of stem state-associated genes were
observed. Among the upregulated genes were a histone H3K9 demethylase (Kdm3a) and a matrix metalloelastase (Mmp12). Upregulation
of these transcripts was dependent upon the transcription factor, hypoxia inducible factor (HIF). We hypothesized that Kdm3a was a
mediator of hypoxia-directed trophoblast cell lineage differentiation and that Mmp12 was a key downstream target responsible for the
trophoblast cell invasive and vascular remodeling phenotype. Consistent with the hypothesis, knockdown of Kdm3a in rat TS cells
inhibited the expression of a subset of the hypoxia/HIF-dependent transcripts, including Mmp12, and altered locus specific as well as
global H3K9 methylation status. Conversely ectopic expression of Kdm3a upregulated a subset of hypoxia/HIF-dependent transcripts,
including Mmp12, and this upregulation was dependent on Kdm3a enzymatic activity. Furthermore, Kdm3a knockdown decreased
hypoxia-induced trophoblast cell invasion in both in vitro and in vivo experiments. Mmp12 possesses the capacity to degrade elastin and
modify the structure of arterial blood vessels. To further explore the functional importance of Mmp12 in trophoblast cell-directed uterine
spiral artery remodeling, we generated an Mmp12 mutant rat model using TALEN-mediated genome editing. A rat model was established
with a 609 bp deletion targeting exon 2 of Mmp12. Homozygous mutant rats showed reduced hypoxia-dependent endovascular trophoblast
invasion and impaired trophoblast-directed uterine spiral artery remodeling. In summary, we have discovered a hypoxia/HIF/Kdm3a
pathway modulating trophoblast cell lineage development, leading to acquisition of the invasive trophoblast cell phenotype, including
upregulation of the extracellular matrix-modifying enzyme Mmp12 and subsequent uterine spiral artery remodeling.
116. Endometrial gene expression in lactating and dry Holstein cows and Holstein heifers on Day 19 of pregnancy.
Stefan Bauersachs1
, Niamh Forde2
, Jochen Bick1
, Stefan Krebs3
, Helmut Blum3
, Eckhard Wolf3
, Patrick Lonergan2
.
1
ETH Zurich, Animal Physiology, Institute of Agricultural Sciences, Zurich, Zurich, Switzerland; 2
School of Agriculture and Food Science,
University College Dublin, Dublin, Leinster, Ireland; 3
Laboratory for Functional Genome Analysis, Gene Center, LMU Munich, Munich,
Bavaria, Germany
The compromised fertility of high producing dairy cows may be caused by negative energy balance. However, the point at which
this metabolic state impacts on impaired fertility is not known. We have previously shown that different types of embryos elicit different
transcriptomic responses from the endometrium. The aim of this study was to test the hypothesis that lactation alters the ability of the
endometrium to respond appropriately to the developing conceptus. Endometrial gene expression on Day 19 of pregnancy in a model of
metabolic stress was analyzed using RNA sequencing. Immediately after calving, primiparous Holstein cows with similar production and
fertility estimated breeding values (EBVs) were randomly divided into two groups, standard lactation (“Lact”) or dried off immediately
(“Dry”, i.e., never milked). Pregnancy was established by transferring grade 1 embryos recovered from superovulated Holstein heifers
(“Heif”) with similar EBVs for production and fertility into lactating and nonlactating recipients (n=1 per recipient). A control group of
Holstein heifers (“Heif”), with similar EBVs for production and fertility as groups 1 and 2, was artificially inseminated. Endometrial tissue
samples were collected after slaughter and recovery of a conceptus on Day 19 of pregnancy. Total RNA was isolated from intercaruncular
endometrium. Strand-specific RNA-Seq libraries (Heif n=4, Lact n=5, Dry n=8) were produced using the Encore Complete RNA-Seq DR
Multiplex System (NuGEN, San Carlos, CA) and run on an Illumina HiSeq 1500. Obtained 100 bp single-end reads (28-65 Mio reads per
sample) were processed using Trimmomatic (version 0.32.1) and mapped to the bovine genome assembly Btau_4.6.1 (bosTau7) with
TopHat2. Mapped reads were counted per gene by the use of QuasR qCount. Statistical analysis with DESeq2 revealed 17 differentially
expressed genes (DEG) between Dry and Heif (FDR 5%) and 135 DEG (FDR 5%) between Lact and Heif. The comparison of Lact and
Dry did not reveal differences at a significance threshold of 5%. Overall, gene expression differences were rather small and lower than two-
fold. Multi-dimensional scaling plots and principal component analysis confirmed the more pronounced differences in gene expression
between lactating cows and heifers and showed that gene expression in the endometrium of dry cows is intermediate compared to the Lact
and Heif groups. Furthermore, higher variation of gene expression in the Dry group was observed for the genes differential between Heif
and Lact. Functional annotation of the DEG between Lact and Heif revealed, e.g., endosome, cytoplasmic vesicle, endocytosis, regulation
of exocytosis, and cytokine receptor activity as overrepresented. The functional categories related to vesicle-mediated transport were
specifically enriched for genes with higher expression in lactating cows and defense response for genes with higher expression in heifers.
In conclusion, our data suggest that parity (cow vs. heifer) and metabolic condition (negative vs. normal energy balance) modulate the
ability of the endometrium to respond to a high-quality embryo on the level of the transcriptome. This research was supported by the
European Union (7th
Framework Program, KBBE.2012.1.3-04: “Optimised terrestrial farm animal reproduction systems and/or
technologies”, project FECUND).