title: Comparison of CD4+ T cell function between HIV-1 resistant and HIV-1 susceptible individuals (Immune)

status: Public on Mar 02 2010

city: Boston

first: Paul

last: McLaren

state: MA

address: 77 Avenue Louis Pasteur, NRB150

department: Medicine

institute: Harvard Medical School

summary:
Understanding why some indidivual resist HIV-1 infection despite continued exposure is an important goal for vaccine development.
We compared CD4+ T cell gene expression at baseline and after antigenic stimulation in HIV-1 resistant commercial sex-workers from Nairobi, Kenya to HIV-1 low-risk negative (non-resistant) non-commercial sex-workers using immune-focused gene expression arrays

Keywords: Case-control, disease state analysis

overall_design: CD4+ T cells from both HIV resistant and HIV low-risk negative individuals were isolated from PBMC after 24 hours of culture by negative slelction. Total RNA was isolated and gene expression compared using immune-focused expression arrays.

first: T

last: Ball

first: Charles

last: Wachihi

first: Walter

last: Jaoko

last: Kelvin

first: Ali

last: Danesh

first: Michael

last: Mayne

first: Larry

middle: D

last: Greller

first: Roland

last: Somogyi

first: Joshua

last: Kimani

first: Francis

last: Plummer

first: Keith

last: Fowke


title: Requirement for Shh and Fox family genes at different stages in sweat gland development (E15.5)

status: Public on Feb 20 2009

city: Baltimore

first: Chang-Yi

last: Cui

state: MD

address: 251 Bayview Blvd Suite 100, 10C222

department: NIA

country: USA

institute: NIH

summary:
Sweat glands play a fundamental role in thermal regulation in man, but the molecular mechanism of their development remains unknown. To initiate analyses, we compared the model of Eda mutant Tabby mice, in which sweat glands were not formed, to wild-type mice.  We inferred developmental stages and critical genes based on observations at 7 time points spanning embryonic, postnatal and adult life. In wild-type footpads, sweat gland germs were detected at E17.5. The coiling of secretory portions started at postnatal day 1 (P1), and sweat gland formation was essentially complete by P5. Consistent with a controlled morphological progression, expression profiling revealed stage-specific gene expression changes. Similar to the development of hair follicles     the other major skin appendage controlled by EDA     sweat gland induction and initial progression was accompanied by Eda-dependent up-regulation of the Shh pathway. During the further development of sweat gland secretory portions, Foxa1 and Foxi1, not at all expressed in hair follicles, were progressively up-regulated in wild-type but not in Tabby footpads. Upon completion of wild-type development, Shh declined to Tabby levels, but Fox family genes remained at elevated levels in mature sweat glands. The results provide a framework for the further analysis of phased downstream regulation of gene action, possibly by a signaling cascade, in response to Eda.

Keywords: cell type comparison design,development or differentiation design

overall_design: To define target genes of Eda during sweat gland development, we  carried out microarray experiments with mouse footpads that from 7  developmental time points including E15.5, E16.5, E17.5, P1, P3, P5 and  8 weeks of wild-type and Tabby mice.  This dataset is E15.5.

first: Makoto

last: Kunisada

first: Yulan

last: Piao

first: Minoru

middle: S

last: Ko

first: David

last: Schlessinger


title: Requirement for Shh and Fox family genes at different stages in sweat gland development (E16.5)

overall_design: To define target genes of Eda during sweat gland development, we  carried out microarray experiments with mouse footpads that from 7  developmental time points including E15.5, E16.5, E17.5, P1, P3, P5 and  8 weeks of wild-type and Tabby mice. This dataset is E16.5.


title: Functional Heterogeneity of Embryonic Stem Cells Revealed Through Translational Amplification of an Early Endodermal Transcript

status: Public on Jun 08 2010

address: 251 Bayview Blvd, Suite 100, 10C

department: National Institute on Aging

summary:
Identification of transcripts in different subpopulations of the HIV mouse ES cell line growing under self-renewing conditions (+Lif, +10FCS, GMEM media and plated on gelatin coated dishes). Subpopulations were identified and isolated based on the expression of the cell surface marker, SSEA 1 (a marker of murine ES cells) and expression of the venus protein, the cDNA of which was knocked into the Hex locus (Hhex). Following growth for 24 hours after initial plating, approximately 10,000,000 cells were dissasociated from dishes with the use of Cell Dissasociating Buffer (Gibco). Cells were resuspended in FACs buffer (10%FCS in PBS) and incubated on ice. Marking of SSEA 1expression was achieved by a 10 minute incubation with  a mouse monoclonal antibody to SSEA 1 (MC-480, Developmental Hybridoma Studies Bank, University of Iowa). Following several washes with FACs buffer, cells were then incubated with an Alexa-647 conjugated anti-mouse IgM anitbody (Invitrogen) for a further 10 minutes. Cells were then washed and resuspended in FACs buffer and subpopulations were fractionated by flow cytometry from two different clones of the HIV cell line (clone 5.1 and clone 16.1). The aim of this array experimnt is to identify significant differences in transcript levels of the four subpopulation from the HIV cell line. Differences of transcript levels from the subpopulations should be consistant among the biological and technical replicates for each.

Keywords: transcript identification design

overall_design: Four subpopulations were collected from clones 5.1 and 16.1 of the HIV cell line as such: V-, S+; V+, S+; V-, S-; V-, S+, where V = Venus expression and S = SSEA 1 expression. RNA was extracted from each subpopulation of each fractionated clone and samples were supplied as duplicates. Therefore each subpopulation is represented as duplicates biologically and as duplicates technically (effectively four times for each). RNA isolation was performed with Trizol reagent according to the manufacturer's instructions. RNA pelletes were resuspended in Nuclease Free Water (ambion) and was measured using the Nanodrop system. 260/280 values for all samples were in the range of 1.8-2.0

first: Maurice

last: Canham

first: Alexei

last: Sharov

middle: M

last: Brickman


title: Database for mRNA Half-Life of 19,977 Genes Obtained by DNA Microarray Analysis of Pluripotent and Differentiating Mouse

status: Public on Nov 15 2008

summary:
Degradation of mRNA is one of the key processes that control the steady-state level of gene expression. However, the rate of mRNA decay for the majority of genes is not known. We successfully obtained the rate of mRNA decay for 19 977 non-redundant genes by microarray analysis of RNA samples obtained from mouse embryonic stem (ES) cells. Median estimated half-life was 7.1 h and only <100 genes, including Prdm1, Myc, Gadd45 g, Foxa2, Hes5 and Trib1, showed half-life less than 1 h. In general, mRNA species with short half-life were enriched among genes with regulatory functions (transcription factors), whereas mRNA species with long half-life were enriched among genes related to metabolism and structure (extracellular matrix, cytoskeleton). The stability of mRNAs correlated more significantly with the structural features of genes than the function of genes: mRNA stability showed the most significant positive correlation with the number of exon junctions per open reading frame length, and negative correlation with the presence of PUF-binding motifs and AU-rich elements in 30 -untranslated region (UTR) and CpG di-nucleotides in the 50 -UTR. The mRNA decay rates presented in this report are the largest data set for mammals and the first for ES cells.

Keywords: growth condition design,reference design,replicate design,RNA stability design,time series design

overall_design: We used two mouse ESC lines: MC1 derived from mouse strain 129S6/SvEvTac and MC2-B6 derived from strain C57BL/6J. To remove contaminating feeder cells, ES cells were first cultured for two passages on gelatin-coated culture dish in complete ES medium. Differentiated ES cells were obtained from MC1 cells by LIF withdrawal (complete medium without LIF, below referenced as LIF-) or by retinoic acid (RA) treatment. To measure mRNA stability, transcription was blocked by adding actinomycin D to the medium. RNA was hybridized in duplicate for each cell type and time point. All hybridizations were carried out by combining a Cy3-CTP-labeled experimental target and a Cy5-CTP-labeled Universal Mouse Reference target.

first: Lioudmila

middle: V

last: SHAROVA

middle: A

last: SHAROV

first: Timur

last: NEDOREZOV

last: PIAO

first: Nabeebi

last: SHAIK

last: KO


title: Defining Developmental Potency and Cell Lineage Trajectories by Expression Profiling of Differentiating Mouse ES Cells

status: Public on Dec 28 2008

summary:
Biologists rely on morphology, function, and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis (PCA) of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three cell lineage trajectories, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast, and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ (EG) cells and induced pluripotent (iPS) cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast (MEF) and fibroblast cell lines are mapped near the far end of the trajectories. We propose that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation.

Keywords: cell type comparison design,reference design,replicate design,time series design

overall_design: Most of the cells and RNA samples used in this study were described in detail previously (See paper's citation associated with this dataset). To maximize the uniformity of the microarray data, all the samples, including ones analyzed by DNA microarray previously, were hybridized to the same platform (the NIA Mouse 44K Microarray  manufactured by Agilent Technologies: AMADID #015087). The intensity of each gene feature per array was extracted from scanned microarray images using Feature Extraction Software V9.5.

first: Kazuhiro

last: Aiba

last: Nedorezov

first: Akira

last: Nishiyama

first: Ryo

last: Matoba

last: Sharova

first: Shinya

last: Yamanaka

first: Hitoshi

last: Niwa


title: Timed induction of 10 transcription factors - ES time series data

status: Public on Oct 01 2009

last: Ko

summary:
To decipher the structure and behaviors of the transcription factor (TF) network, we created 50 permanent mouse ES cell lines, in each of which one of the 50 transcription factors tagged with FLAG, is inserted into the doxycycline (dox)-repressible ROSA26 locus. We have obtained time series data for 10 of the transcripts at 24h,48h and 72hr. Expression profiling reveals Cdx2 as the most potent inducer of transcriptome perturbation in ES cells, followed by Esx1, Sox9, Tcf3, Klf4, and Gata3. Immunoprecipitation (IP) with a FLAG-antibody in Cdx2-induced ES cells, identifies NuRD in CDX2-associated protein complexes; and chromatin-IP-sequencing identifies CDX2-binding sites predominantly in genes up-regulated by CDX2. Compendium analyses of Cdx2- and the other TF-inducible ES cells suggest a central role of the POU5F1/SOX2/NANOG protein complex in a swift-acting control mechanism to down-regulate a common set of genes at the beginning of multi-lineage ES cell differentiations. These ES cell lines will be a valuable resource to study biological networks in ES cells and mice.

Keywords: dose response design,genetic modification design,individual genetic characteristic design,reference design,replicate design,time series design

overall_design: MC1 mouse ES cells derived from 129S6/SvEvTac were cultured in DMEM with 15% FBS and LIF on feeder cells. Cells were electroporated with linearlized pMWROSATcH and selected by hygromycine B. Knock-in for ROSA-TET locus in ES[MC1R(20)] cells was confirmed by southern blotting. For exchange vectors, PCR amplified ORFs were subcloned into pZhcSfi that was modified to express His6-FLAG tagged protein and puromycin resistant gene. ES[MC1R(20)] cells (passage 17) cultured on feeder cells were co-transfected with sequence verified exchange vector and pCAGGS-Cre and selected by puromycin in the presence of doxycycline. Isolated clones were tested for Venus expression, hygromycin B susceptibility, transgene RNA expression, genotyping for Cre mediated integration, karyotyping, western blotting using anti-FLAG antibody (Sigma-Aldrich) and mycoplasma contamination. ES cells (passage 25) were plated onto gelatin-coated dish. Doxycycline was removed through washing 3 times with PBS at 3 hours intervals. Total RNA was isolated by TRIzol (Invitrogen) at time points indicated in the sample names. Cy3-CTP labeled sample targets were prepared with total RNA by Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Cy5-CTP labeled reference target was produced from mixture of Stratagene Universal Mouse Reference RNA and MC1 cells RNA.


title: Timed induction of 50 transcription factors in ES cells reveals a common mechanism to initiate differentiations

summary:
To decipher the structure and behaviors of the transcription factor (TF) network, we created 50 permanent mouse ES cell lines, in each of which one of the 50 transcription factors tagged with FLAG, is inserted into the doxycycline (dox)-repressible ROSA26 locus. Expression profiling reveals Cdx2 as the most potent inducer of transcriptome perturbation in ES cells, followed by Esx1, Sox9, Tcf3, Klf4, and Gata3. Immunoprecipitation (IP) with a FLAG-antibody in Cdx2-induced ES cells, identifies NuRD in CDX2-associated protein complexes; and chromatin-IP-sequencing identifies CDX2-binding sites predominantly in genes up-regulated by CDX2. Compendium analyses of Cdx2- and the other TF-inducible ES cells suggest a central role of the POU5F1/SOX2/NANOG protein complex in a swift-acting control mechanism to down-regulate a common set of genes at the beginning of multi-lineage ES cell differentiations. These ES cell lines will be a valuable resource to study biological networks in ES cells and mice.

Keywords: dose response design,genetic modification design,individual genetic characteristic design,reference design,replicate design

overall_design: MC1 mouse ES cells derived from 129S6/SvEvTac were cultured in DMEM with 15% FBS and LIF on feeder cells. Cells were electroporated with linearlized pMWROSATcH and selected by hygromycine B. Knock-in for ROSA-TET locus in ES[MC1R(20)] cells was confirmed by southern blotting. For exchange vectors, PCR amplified ORFs were subcloned into pZhcSfi that was modified to express His6-FLAG tagged protein and puromycin resistant gene. ES[MC1R(20)] cells (passage 17) cultured on feeder cells were co-transfected with sequence verified exchange vector and pCAGGS-Cre and selected by puromycin in the presence of doxycycline. Isolated clones were tested for Venus expression, hygromycin B susceptibility, transgene RNA expression, genotyping for Cre mediated integration, karyotyping, western blotting using anti-FLAG antibody (Sigma-Aldrich) and mycoplasma contamination. ES cells (passage 25) were plated onto gelatin-coated dish. Doxycycline was removed through washing 3 times with PBS at 3 hours intervals. Total RNA was isolated by TRIzol (Invitrogen) after 2 days. Cy3-CTP labeled sample targets were prepared with total RNA by Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Cy5-CTP labeled reference target was produced from mixture of Stratagene Universal Mouse Reference RNA and MC1 cells RNA.

first: Li

last: Xin

first: Marshall

middle: P

last: Thomas

first: Gregory

last: Mowrer

first: Emily

last: Meyers

last: Piao

first: Samir

last: Mehta

first: Sarah

last: Yee

first: Carole

last: Stagg

first: Lina

last: Correa-Cerro

first: Uwem

last: Bassey

first: Yuhki

last: Nakatake

first: Hien

last: Hoang

first: Eugene

last: Kim

first: Richard

last: Tapnio

first: Yong

last: Qian

first: Dawood

last: Dudekula

first: Michal

last: Zalzman

first: Manxiang

last: Li

first: Geppino

last: Falco

first: Hsih-Te

last: Yang

first: Sung-Lim

last: Lee

first: Manuela

last: Monti

first: Ilaria

last: Stanghellini

first: Md

middle: N

last: Islam

first: Ramaiah

last: Nagaraja

first: Ilya

last: Goldberg

first: Weidong

last: Wang

first: Dan

middle: L

last: Longo

last: Schlessinger