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Reprogramming to pluripotency through a somatic stem cell intermediate

ABSTRACT: In the past years, transdifferentiation from one lineage to another has become an important research field. In that context, we recently reported the direct reprogramming of mouse embryonic fibroblasts (MEFs) into induced neural stem cells (iNSCs). In this study, we, for the first time, successfully reprogrammed these iNSCs into induced pluripotent stem cells (iPSCs), which we termed iNSC-derived iPSCs (iNdiPSCs). iNdiPSCs proved to be truly pluripotent, as shown by a pluripotent gene expression profile and the ability to differentiate into all three germ layers both, in vitro and in vivo. We could further show that iNdiPSCs do not retain an epigenetic memory neither from iNSCs nor MEFs. In conclusion, our data validate the stable somatic stem cell status of iNSCs and demonstrate their potential to give rise to bona fide iPSCs that are indistinguishable from other iPSCs or embryonic stem cells. Cell culture conditions: iNdiPSCs were grown on feeder cells in 2i medium (Knockout DMEM supplemented with 20% Knockout serum replacement, 0.2x -Mercaptoethanol (all GIBCO), 1x nonessential amino acids and 1x penicillin/streptomycin/glutamine (PSG) (both PAA), 1nM PD0325901, 3nM CHIR99021 (both Axon Medchem), and 1000 U/ml leukemia inhibitory factor [in house preparation]). Virus production and infection: Commercially available pMX retroviruses expressing mouse Oct4 (Addgene # 13366) and Klf4 (# 13370) were transfected in 293T cells in iNSC medium: DMEM/F-12 supplemented with B27 (both GIBCO), 10ng/ml EGF, 10ng/ml bFGF (both Peprotech) and 1x PSG. Supernatant was collected after 48 h, filtered and used for infection of 5 x 104 iNSCs. After 48 h cells were cultured in 2i medium. Alkaline phosphatase staining Cells were fixed for 5 min in 4% paraformaldehyde (PFA) at room temperature (RT), washed twice with phosphate buffered saline (PBS) (PAA) and stained with the SIGMA FAST Fast Red TR/Napthol AS-MX kit (product no. F4648) for 20 min in the dark at RT. Chromosome spread: Cells were subjected to 100ng/ml KaryoMax Colcemid (GIBCO) for 3 h at 37C. Cells were harvested and resuspended by slowly adding 4 ml 0.56% prewarmed KCl, incubated for 15 min at 37C and spun down at 900 rpm for 5 min. Fresh fixative (3 parts methanol plus 1 part acetic acid) was very slowly added to the pellet to a final volume of 3 ml and incubated for 30 min at RT. Cells were subsequently washed 3 times in fixative and resuspened in fixative. Approximately 20 microl were dropped from a height of about 2 m on a dry slide. Drops were air dried for 30 min and mounted with DAPI containing mounting solution. RNA extraction, cDNA synthesis and quantitative real time PCR (qRTPCR) RNA was extracted using the QIAGEN RNeas Mini Kit (cat no. 74106) and cDNA synthesis performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRTPCR was carried out using iTaq SYBR Green Supermix With ROX (Bio-Rad). Oct4 promoter methylation analysis: Bisulfite conversion was performed using the QIAGEN EpiTectﾮ Bisulfite Kit (Cat no. 59104). Followed by nested PCR using a set of target region specific primers. The first PCR was performed using 1micro-l of bisulfite converted and purified DNA, SuperTaq polymerase (Promega) according to the manufacturers protocol and a total of 35 cycles with denaturation at 94C for 30 s, followed by 30 s of annealing at 50C and 72C elongation for 1 min with an initial denaturation at 94C and a final elongation step at 72C for 3 min each. For the second PCR, we used Taq polymerase according to the manufacturers protocol (New England Biolabs) and 1 microl of the first PCR product with denaturation at 95C for 30 s, followed by 40 cycles of denaturation for 30 s at 95C, annealing for 30 s at 55C and elongation for 1 min at 68C, and finally an additional elongation step for 5 min at 68C. The amplified fragments were resolved on a 1.5% agarose gel, purified using the QIAGEN QIAquick Gel Extraction Kit (Cat no. 28706) and subsequently cloned into the pCRII-TOPO-vector using the TOPO TA Cloning Kit (Invitrogen). The cloned products were transformed into One Shot TOP10 competent bacteria (Invitrogen) and plated on agar plates. The picked colonies were sent to GATC for sequencing. The results were then analyzed with the QUMA software (online available at www.quma.cdb.riken.jp). Immunocytochemistry: Cells were fixed for 5 min in 4% PFA at RT, washed twice in PBS, permeabilized with 0.1% Triton X-100 for 10 min, and washed again twice in PBS. Followed by blocking with 5% bovine serum albumin (BSA) (GIBCO) for 1 h shaking at RT. Subsequently the first antibody was added in 1% BSA and incubated for 1 h shaking at RT. The cells were again washed twice in PBS and subjected to the second antibody in 1% BSA for 1 h shaking at RT and then washed three times with PBS, including DAPI in the second wash. The following primary antibodies were used: anti-Nanog (REC-RCAB0002P-F, Cosmo Bio Co.) 1:1000 anti SSEA1, (MC-480, Millipore), 1:160, anti-3-Tubulin (T8660, Sigma), 1:2000, anti-Smooth muscle actin (M0851, DAKO), 1:200, anti-Sox17 (AF1924, R&D Systems), 1:100. The following secondary antibodies were used: Alexa Fluor 568 goat anti-rabbit, rabbit anti-mouse, and rabbit anti-goat (Invitrogen), all 1:2000. DNA extraction and PCR: DNA extraction was performed using the QIAGEN QIAampﾮ DNA Mini Kit (cat no. 51306). PCR was carried out using Taq polymerase including buffers from New England Biolabs, following their recommended protocol. The following primers were used for genotyping: pMX-Oct4, -Klf4, -Sox2, -c-Myc and -Brn4. In vitro differentiation: First, hanging drops were formed placing 20ﾵl drops containing 600 cells each on the lid of a 10cm cell culture dish, which was then inverted. After 3 days, hanging drops were transferred to gelatin-coated dishes and cultured in standard MEF medium (DMEM containing 20% fetal calf serum (both GIBCO) and 1x PSG), which was also used for hanging drop formation. After approximately 10-14 days the cells were fixed and stained as described above. Teratoma formation: 1 x 106 iNdiPSCs were injected subcutaneously into SCID mice. Tumors were resected after 4 weeks and fixed in 4% PFA. Paraffin sections were cut, mounted on glass slides and stained with haematoxylin and eosin. Animal handling was performed in accordance with the German animal protection law and the guidelines of the Max Planck Institute. Microarray analysis: Purified cRNA samples were prepared with the linear TotalPrep RNA Amplification Kit (Ambion) from 500 ng original RNA, involving synthesis of double-stranded cDNA by T7-promoter-linked oligo(dT) primers and 14 h of in vitro transcription incorporating biotin-labeled nucleotides. Hybridization onto MouseRef 8 v2.0 Expression BeadChips (Illumina) was performed according to the manufacturerﾒs recommendations. Chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader (Illumina). The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix robust multiarray analysis (RMA) background correction model. Variance stabilization was performed by log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor.

ORGANISM(S): Mus musculus

PROVIDER: GSE44284 | GEO | 2014/03/28

SECONDARY ACCESSION(S): PRJNA189293

REPOSITORIES: GEO

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Project description:In the past years, transdifferentiation from one lineage to another has become an important research field. In that context, we recently reported the direct reprogramming of mouse embryonic fibroblasts (MEFs) into induced neural stem cells (iNSCs). In this study, we, for the first time, successfully reprogrammed these iNSCs into induced pluripotent stem cells (iPSCs), which we termed iNSC-derived iPSCs (iNdiPSCs). iNdiPSCs proved to be truly pluripotent, as shown by a pluripotent gene expression profile and the ability to differentiate into all three germ layers both, in vitro and in vivo. We could further show that iNdiPSCs do not retain an epigenetic memory neither from iNSCs nor MEFs. In conclusion, our data validate the stable somatic stem cell status of iNSCs and demonstrate their potential to give rise to bona fide iPSCs that are indistinguishable from other iPSCs or embryonic stem cells. Cell culture conditions: iNdiPSCs were grown on feeder cells in 2i medium (Knockout DMEM supplemented with 20% Knockout serum replacement, 0.2x -Mercaptoethanol (all GIBCO), 1x nonessential amino acids and 1x penicillin/streptomycin/glutamine (PSG) (both PAA), 1nM PD0325901, 3nM CHIR99021 (both Axon Medchem), and 1000 U/ml leukemia inhibitory factor [in house preparation]). Virus production and infection: Commercially available pMX retroviruses expressing mouse Oct4 (Addgene # 13366) and Klf4 (# 13370) were transfected in 293T cells in iNSC medium: DMEM/F-12 supplemented with B27 (both GIBCO), 10ng/ml EGF, 10ng/ml bFGF (both Peprotech) and 1x PSG. Supernatant was collected after 48 h, filtered and used for infection of 5 x 104 iNSCs. After 48 h cells were cultured in 2i medium. Alkaline phosphatase staining Cells were fixed for 5 min in 4% paraformaldehyde (PFA) at room temperature (RT), washed twice with phosphate buffered saline (PBS) (PAA) and stained with the SIGMA FAST Fast Red TR/Napthol AS-MX kit (product no. F4648) for 20 min in the dark at RT. Chromosome spread: Cells were subjected to 100ng/ml KaryoMax Colcemid (GIBCO) for 3 h at 37C. Cells were harvested and resuspended by slowly adding 4 ml 0.56% prewarmed KCl, incubated for 15 min at 37C and spun down at 900 rpm for 5 min. Fresh fixative (3 parts methanol plus 1 part acetic acid) was very slowly added to the pellet to a final volume of 3 ml and incubated for 30 min at RT. Cells were subsequently washed 3 times in fixative and resuspened in fixative. Approximately 20 microl were dropped from a height of about 2 m on a dry slide. Drops were air dried for 30 min and mounted with DAPI containing mounting solution. RNA extraction, cDNA synthesis and quantitative real time PCR (qRTPCR) RNA was extracted using the QIAGEN RNeas Mini Kit (cat no. 74106) and cDNA synthesis performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRTPCR was carried out using iTaq SYBR Green Supermix With ROX (Bio-Rad). Oct4 promoter methylation analysis: Bisulfite conversion was performed using the QIAGEN EpiTectﾮ Bisulfite Kit (Cat no. 59104). Followed by nested PCR using a set of target region specific primers. The first PCR was performed using 1micro-l of bisulfite converted and purified DNA, SuperTaq polymerase (Promega) according to the manufacturers protocol and a total of 35 cycles with denaturation at 94C for 30 s, followed by 30 s of annealing at 50C and 72C elongation for 1 min with an initial denaturation at 94C and a final elongation step at 72C for 3 min each. For the second PCR, we used Taq polymerase according to the manufacturers protocol (New England Biolabs) and 1 microl of the first PCR product with denaturation at 95C for 30 s, followed by 40 cycles of denaturation for 30 s at 95C, annealing for 30 s at 55C and elongation for 1 min at 68C, and finally an additional elongation step for 5 min at 68C. The amplified fragments were resolved on a 1.5% agarose gel, purified using the QIAGEN QIAquick Gel Extraction Kit (Cat no. 28706) and subsequently cloned into the pCRII-TOPO-vector using the TOPO TA Cloning Kit (Invitrogen). The cloned products were transformed into One Shot TOP10 competent bacteria (Invitrogen) and plated on agar plates. The picked colonies were sent to GATC for sequencing. The results were then analyzed with the QUMA software (online available at www.quma.cdb.riken.jp). Immunocytochemistry: Cells were fixed for 5 min in 4% PFA at RT, washed twice in PBS, permeabilized with 0.1% Triton X-100 for 10 min, and washed again twice in PBS. Followed by blocking with 5% bovine serum albumin (BSA) (GIBCO) for 1 h shaking at RT. Subsequently the first antibody was added in 1% BSA and incubated for 1 h shaking at RT. The cells were again washed twice in PBS and subjected to the second antibody in 1% BSA for 1 h shaking at RT and then washed three times with PBS, including DAPI in the second wash. The following primary antibodies were used: anti-Nanog (REC-RCAB0002P-F, Cosmo Bio Co.) 1:1000 anti SSEA1, (MC-480, Millipore), 1:160, anti-3-Tubulin (T8660, Sigma), 1:2000, anti-Smooth muscle actin (M0851, DAKO), 1:200, anti-Sox17 (AF1924, R&D Systems), 1:100. The following secondary antibodies were used: Alexa Fluor 568 goat anti-rabbit, rabbit anti-mouse, and rabbit anti-goat (Invitrogen), all 1:2000. DNA extraction and PCR: DNA extraction was performed using the QIAGEN QIAampﾮ DNA Mini Kit (cat no. 51306). PCR was carried out using Taq polymerase including buffers from New England Biolabs, following their recommended protocol. The following primers were used for genotyping: pMX-Oct4, -Klf4, -Sox2, -c-Myc and -Brn4. In vitro differentiation: First, hanging drops were formed placing 20ﾵl drops containing 600 cells each on the lid of a 10cm cell culture dish, which was then inverted. After 3 days, hanging drops were transferred to gelatin-coated dishes and cultured in standard MEF medium (DMEM containing 20% fetal calf serum (both GIBCO) and 1x PSG), which was also used for hanging drop formation. After approximately 10-14 days the cells were fixed and stained as described above. Teratoma formation: 1 x 106 iNdiPSCs were injected subcutaneously into SCID mice. Tumors were resected after 4 weeks and fixed in 4% PFA. Paraffin sections were cut, mounted on glass slides and stained with haematoxylin and eosin. Animal handling was performed in accordance with the German animal protection law and the guidelines of the Max Planck Institute. Microarray analysis: Purified cRNA samples were prepared with the linear TotalPrep RNA Amplification Kit (Ambion) from 500 ng original RNA, involving synthesis of double-stranded cDNA by T7-promoter-linked oligo(dT) primers and 14 h of in vitro transcription incorporating biotin-labeled nucleotides. Hybridization onto MouseRef 8 v2.0 Expression BeadChips (Illumina) was performed according to the manufacturerﾒs recommendations. Chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader (Illumina). The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix robust multiarray analysis (RMA) background correction model. Variance stabilization was performed by log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. 8 samples were analyzed. MEF: Regular mouse embryonic fibroblasts with Tet-On background, 1 replicate NSC: Regular mouse neural stems cells with Tet-On background, 1 replicate iNSC: mouse induced neural stems cells, 1 replicate iNdiPSC-1: mouse induced pluripotent cells (IPSs) derived from iNSC (clon 1), 1 replicate iNdiPSC-4: mouse induced pluripotent cells (IPSs) derived from iNSC (clon 4), 1 replicate NSC2FiPSC: mouse induced pluripotent cells (IPSs) derived from regular NSC (using reprogramming 2 factors), 1 replicate MEF4FiPSC: mouse induced pluripotent cells (IPSs) derived from regular MEF (using reprogramming 4 factors), 1 replicate ESC: regular mouse embryonic stem cells with Tet-On background, 1 replicate

Project description:U2OS osteosarcoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) and Raji B-cells in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS, Bio&Sell), 2 mM L-glutamine (Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco) at 37°C at 8% or 5% CO2, respectively. Chromatin immunoprecipitation for ChIP-seq: Cells were crosslinked using a formaldehyde containing solution (10mM NaCl, 0,1mM EDTA pH 8, 0,05mM EGTA pH 8, 5mM HEPES pH 7.8 and 1% formaldehyde) for 10 minutes at 20°C, the reaction was quenched by the addition of glycin to a final concentration of 250uM for 5 minutes. Crosslinked cells were collected and washed twice with PBS before snap freezing in liquid nitrogen and storage at -80°C until subsequent use. Prior to sonication, the crosslinked cells were resuspended in lysis buffer (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100, 1X protease inhibitor cocktail) at 4°C for 20 minutes. Nuclei were collected by centrifugation and washed in a second buffer (200mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 1X protease inhibitor cocktail) for 10 minutes at 4°C then collected by centrifugation and resuspended in the shearing buffer (1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 100mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1X protease inhibitor cocktail). Sonication was carried out in a Bioruptor Pico ultrasounds water bath (Diagenode B01060001) for 30 cycles of 30 sec ON and 30 sec OFF pulses in 4°C water. Sonicated extracts were centrifuged at high speed in the presence of 0,1% of Triton X-100 and snap frozen in liquid nitrogen then stored at -80°C until subsequent use. Prior to ChIP, the ZNF768 mAb was coupled to protein-G coated magnetic beads (Dynabeads, life technologies) by incubation in 0,5% BSA PBS overnight at 4°C. Pre-coated beads were then washed and incubated with the sonicated chromatin extracts, the ChIP was carried out overnight at 4°C on a rotating wheel. The equivalent of 10 × 107 cells sonicated extract was used for each ZNF ChIP experiment for both cell lines. After incubation, the beads were washed 7 times with Wash buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA pH 8, 1% NP-40, 0.7% Na-Deoxycholate, 1X protease inhibitor cocktail) followed by one wash with TE-NaCl buffer (10mM Tris pH 8 and 1mM EDTA pH 8, 50mM NaCl). Immunoprecipitated chromatine was eluted by two sequential incubations with 100µl Elution buffer (50mM Tris pH 8, 10mM EDTA pH 8, 1% SDS) at 65°C for 15 minutes. The two eluates were pooled and incubated at 65°C for 12 hours to reverse-crosslink the chromatin followed by treatment with RNase A (0.2µg/mL) at 37°C for two hours and Proteinase K (0.2µg/mL) at 55°C for two hours. The DNA was isolated by phenol:chloroform: isoamylalcohol (25:24:1 pH 8) extranction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). At least 1ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol. The ChIP DNA was size selected using Ampure beads (Life technologies) to enrich for fragments < 400Bp prior to end-repair, 3’end adenylation and adapter ligation. Library fragments were then directly amplified by 10 cycles of PCR.

Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon head kidney. Head kidney mRNA samples were prepared from pooled (n = 10) infected or control kidney. Approximately 200 ng of infected or control head kidney mRNA was used as template in aRNA synthesis reactions. Two infected head kidney samples were pooled to yield 8.9 ug of aRNA, and a single control head kidney sample contained 8.4 ug of aRNA. Infected and control head kidney aRNA samples were visualized on agarose gels to check quality and quantity (data not shown). Except for the amounts of aRNA and reverse transcription (RT) primer used, head kidney target labeling reactions and microarray hybridizations were identical. Head kidney target syntheses used 2 ug of aRNA and 2 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C for 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 75 Cy3, PMT 63 or 64 Cy5 for head kidney study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other

Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon macrophages. Infected and control (24 h incubation) macrophage mRNA samples were subjected to two rounds of amplification using the MessageAmp aRNA kit (Ambion) and manufacturer's instructions. Approximately 400 ng of macrophage mRNA was used as template in the first round of amplification, yielding 8.1 ug of infected macrophage aRNA and 7.8 ug of control macrophage aRNA. Approximately 1 ug of first-round macrophage aRNA was used as template in the second round of amplification, yielding 81.2 ug of infected macrophage aRNA and 83.3 ug of control macrophage aRNA. Infected and control macrophage aRNA samples were visualized on agarose gels to check quality and quantity. Except for the amounts of aRNA and reverse transcription (RT) primer used, macrophage target labeling reactions and microarray hybridizations were identical. Macrophage target syntheses used 5 ug of second-round aRNA and 5 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5'T18[Q-N]3'), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 72 Cy3, PMT 63 or 64 Cy5 for macrophage study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other

Project description:Human hepatocellular carcinoma cells, HepG2 , cultured in DMEM containing 10% fetal calf serum. To study gene expression changes induced by Vitamin K2, total RNA was isolated following the Isogen procedure (Nippon-gene, Tokyo, Japan) from HepG2 cells with and without 50 microM of Vitamin K2 treatment for three days. The cDNA microarrays Human Oligo Chip 30K “AceGene” subset A was purchased from Hitachi Software Engineering Co. (Yokohama-City, Japan). This array contains 10368 kinds of gene-specific 50 mer sense oligonucleotides. Subset A contains mainly known function ORF oligo probes. Preparation of fluorescent cDNA targets by an indirect labeling approach was performed using PowerScript reverse transcription kit (Clontech). First-strand cDNAs were synthesized from 30 microg of total RNA from HepG2 cells treated with or without Vitamin K2 using random primer and PoweScript Fluorescent Labelling Kit. Monofunctional, N-hydroxysuccinimide-activated fluorescent dyes (Cy3 and Cy5; Amersham) were coupled to the cDNAs by reaction with the amino functional groups. Untreated cells were labeled with Cy3 as a control and treated cells with Cy5 as a sample. After free dyes were removed using MinElute Purification kit (Qiagen), the targets were mixed together and added to the microarrays, and then incubated overnight (16 hours) at 42?C. The slides were washed at 30?C in each with 2x SSC, 0.1% SDS and 2x SSC for 10 min and 5 min, respectively, and washed in 1x SSC for 5 min. Fluorescent images of the hybridized microarrays were scanned with a fluorescence laser confocal slide scanner (Affymetrix 428 Array Scanner, Affymetrix, Santa Clara, CA). The Cy3 and Cy5 intensities with background subtraction were determined by ImaGene 4.2 software (BioDiscovery, Marina Del Rey, CA). The expression data were then filtered based on their channel intensities, spot size and flag (missing or inaccurate data), and the Cy5/Cy3 ratios were calculated and normalized by median-centering the log-ratio of all genes.

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Reprogramming to pluripotency through a somatic stem cell intermediate

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