doxycycline inducible ptrip vectors  (Thermo Fisher)


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    Structured Review

    Thermo Fisher doxycycline inducible ptrip vectors
    Activation of SMAD signaling at low cell density and contribution of ROS signaling. ( A ) <t>Smad7</t> overexpression changes the gene expression profile of LNCaP* cells under LD_hypo conditions. Ratios of mRNA species levels measured by RT-qPCR in <t>pTRIP-mSMAD7</t> and pTRIP-control transduced cell populations cultured under LD_hypo conditions were averaged from 2 experiments using independent cell populations. Stars indicate significance of the decreased mRNA species accumulation upon murine Smad7 overexpression. ( B ) Glutathione supplementation reduces the activation of SMAD signaling at low density. Relative mRNA levels measured by RT-qPCR under LD_hypo, LD_hypo+glutathione and MD_hypo conditions were averaged from 4–5, 2 and 7–9 cell culture experiments, respectively. Stars indicate statistical significance of the difference in mRNA levels between cells cultured in LD_hypo and LD_hypo+glutathione. ( C ) Lack of additive effects between Smad7 overexpression and glutathione treatment on cloning efficiency. Cloning efficiency of pTRIP-control and pTRIP-m Smad 7 transduced cell populations were measured under the indicated cell culture conditions. Data were averaged from 2 to 5 experiments made with 2 independent control and 2 independent Smad7 transduced cell populations processed in parallel. Stars indicate significance of the differences in cloning efficiency. Data are presented as mean ± s.d.
    Doxycycline Inducible Ptrip Vectors, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    doxycycline inducible ptrip vectors - by Bioz Stars, 2020-04
    88/100 stars

    Images

    1) Product Images from "SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy"

    Article Title: SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy

    Journal: Cell Cycle

    doi: 10.1080/15384101.2015.1014145

    Activation of SMAD signaling at low cell density and contribution of ROS signaling. ( A ) Smad7 overexpression changes the gene expression profile of LNCaP* cells under LD_hypo conditions. Ratios of mRNA species levels measured by RT-qPCR in pTRIP-mSMAD7 and pTRIP-control transduced cell populations cultured under LD_hypo conditions were averaged from 2 experiments using independent cell populations. Stars indicate significance of the decreased mRNA species accumulation upon murine Smad7 overexpression. ( B ) Glutathione supplementation reduces the activation of SMAD signaling at low density. Relative mRNA levels measured by RT-qPCR under LD_hypo, LD_hypo+glutathione and MD_hypo conditions were averaged from 4–5, 2 and 7–9 cell culture experiments, respectively. Stars indicate statistical significance of the difference in mRNA levels between cells cultured in LD_hypo and LD_hypo+glutathione. ( C ) Lack of additive effects between Smad7 overexpression and glutathione treatment on cloning efficiency. Cloning efficiency of pTRIP-control and pTRIP-m Smad 7 transduced cell populations were measured under the indicated cell culture conditions. Data were averaged from 2 to 5 experiments made with 2 independent control and 2 independent Smad7 transduced cell populations processed in parallel. Stars indicate significance of the differences in cloning efficiency. Data are presented as mean ± s.d.
    Figure Legend Snippet: Activation of SMAD signaling at low cell density and contribution of ROS signaling. ( A ) Smad7 overexpression changes the gene expression profile of LNCaP* cells under LD_hypo conditions. Ratios of mRNA species levels measured by RT-qPCR in pTRIP-mSMAD7 and pTRIP-control transduced cell populations cultured under LD_hypo conditions were averaged from 2 experiments using independent cell populations. Stars indicate significance of the decreased mRNA species accumulation upon murine Smad7 overexpression. ( B ) Glutathione supplementation reduces the activation of SMAD signaling at low density. Relative mRNA levels measured by RT-qPCR under LD_hypo, LD_hypo+glutathione and MD_hypo conditions were averaged from 4–5, 2 and 7–9 cell culture experiments, respectively. Stars indicate statistical significance of the difference in mRNA levels between cells cultured in LD_hypo and LD_hypo+glutathione. ( C ) Lack of additive effects between Smad7 overexpression and glutathione treatment on cloning efficiency. Cloning efficiency of pTRIP-control and pTRIP-m Smad 7 transduced cell populations were measured under the indicated cell culture conditions. Data were averaged from 2 to 5 experiments made with 2 independent control and 2 independent Smad7 transduced cell populations processed in parallel. Stars indicate significance of the differences in cloning efficiency. Data are presented as mean ± s.d.

    Techniques Used: Activation Assay, Over Expression, Expressing, Quantitative RT-PCR, Cell Culture, Clone Assay

    2) Product Images from "SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy"

    Article Title: SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy

    Journal: Cell Cycle

    doi: 10.1080/15384101.2015.1014145

    Activation of SMAD signaling at low cell density and contribution of ROS signaling. ( A ) Smad7 overexpression changes the gene expression profile of LNCaP* cells under LD_hypo conditions. Ratios of mRNA species levels measured by RT-qPCR in pTRIP-mSMAD7 and pTRIP-control transduced cell populations cultured under LD_hypo conditions were averaged from 2 experiments using independent cell populations. Stars indicate significance of the decreased mRNA species accumulation upon murine Smad7 overexpression. ( B ) Glutathione supplementation reduces the activation of SMAD signaling at low density. Relative mRNA levels measured by RT-qPCR under LD_hypo, LD_hypo+glutathione and MD_hypo conditions were averaged from 4–5, 2 and 7–9 cell culture experiments, respectively. Stars indicate statistical significance of the difference in mRNA levels between cells cultured in LD_hypo and LD_hypo+glutathione. ( C ) Lack of additive effects between Smad7 overexpression and glutathione treatment on cloning efficiency. Cloning efficiency of pTRIP-control and pTRIP-m Smad 7 transduced cell populations were measured under the indicated cell culture conditions. Data were averaged from 2 to 5 experiments made with 2 independent control and 2 independent Smad7 transduced cell populations processed in parallel. Stars indicate significance of the differences in cloning efficiency. Data are presented as mean ± s.d.
    Figure Legend Snippet: Activation of SMAD signaling at low cell density and contribution of ROS signaling. ( A ) Smad7 overexpression changes the gene expression profile of LNCaP* cells under LD_hypo conditions. Ratios of mRNA species levels measured by RT-qPCR in pTRIP-mSMAD7 and pTRIP-control transduced cell populations cultured under LD_hypo conditions were averaged from 2 experiments using independent cell populations. Stars indicate significance of the decreased mRNA species accumulation upon murine Smad7 overexpression. ( B ) Glutathione supplementation reduces the activation of SMAD signaling at low density. Relative mRNA levels measured by RT-qPCR under LD_hypo, LD_hypo+glutathione and MD_hypo conditions were averaged from 4–5, 2 and 7–9 cell culture experiments, respectively. Stars indicate statistical significance of the difference in mRNA levels between cells cultured in LD_hypo and LD_hypo+glutathione. ( C ) Lack of additive effects between Smad7 overexpression and glutathione treatment on cloning efficiency. Cloning efficiency of pTRIP-control and pTRIP-m Smad 7 transduced cell populations were measured under the indicated cell culture conditions. Data were averaged from 2 to 5 experiments made with 2 independent control and 2 independent Smad7 transduced cell populations processed in parallel. Stars indicate significance of the differences in cloning efficiency. Data are presented as mean ± s.d.

    Techniques Used: Activation Assay, Over Expression, Expressing, Quantitative RT-PCR, Cell Culture, Clone Assay

    3) Product Images from "p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition"

    Article Title: p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition

    Journal: eLife

    doi: 10.7554/eLife.01856

    p97 is required for processing and activation of Nrf1. ( A ) NIH-3T3-p97sh cells stably expressing doxycycline (Doxy)-inducible shRNA targeting p97 were either mock-treated or induced with Doxy (1 µg/ml) for 3 days after which the cells were further treated with DMSO or 1 µM MG132 as indicated for 10 hr. The RNA from these cells was used for quantitative RT-PCR to assess the mRNA levels of the representative PSM genes. The values were normalized to GAPDH mRNA levels. Error bars denote SD (n = 3). ( B ) NIH-3T3-p97sh cells were subjected to Doxy and MG132 treatments as above and the cell lysates were fractionated by SDS-PAGE and immunoblotted to detect p97 and Nrf1. β-actin was used as a loading control. ( C ) NIH-3T3 (left panel) or HEK-293-Nrf1 3×Flag cells (right panel) were pulsed for an hour with 10 µM NMS-873 and then chased with MG132 plus cycloheximide (CHX). The cells were harvested at the time points indicated (from 0 min to 120 min) and the lysates were fractionated by SDS-PAGE and immunoblotted to detect endogenous Nrf1. β-actin was used as a loading control. DOI: http://dx.doi.org/10.7554/eLife.01856.005
    Figure Legend Snippet: p97 is required for processing and activation of Nrf1. ( A ) NIH-3T3-p97sh cells stably expressing doxycycline (Doxy)-inducible shRNA targeting p97 were either mock-treated or induced with Doxy (1 µg/ml) for 3 days after which the cells were further treated with DMSO or 1 µM MG132 as indicated for 10 hr. The RNA from these cells was used for quantitative RT-PCR to assess the mRNA levels of the representative PSM genes. The values were normalized to GAPDH mRNA levels. Error bars denote SD (n = 3). ( B ) NIH-3T3-p97sh cells were subjected to Doxy and MG132 treatments as above and the cell lysates were fractionated by SDS-PAGE and immunoblotted to detect p97 and Nrf1. β-actin was used as a loading control. ( C ) NIH-3T3 (left panel) or HEK-293-Nrf1 3×Flag cells (right panel) were pulsed for an hour with 10 µM NMS-873 and then chased with MG132 plus cycloheximide (CHX). The cells were harvested at the time points indicated (from 0 min to 120 min) and the lysates were fractionated by SDS-PAGE and immunoblotted to detect endogenous Nrf1. β-actin was used as a loading control. DOI: http://dx.doi.org/10.7554/eLife.01856.005

    Techniques Used: Activation Assay, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, SDS Page

    Related Articles

    Transduction:

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    Clone Assay:

    Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation
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    Article Title: Protein aggregation of SERCA2 mutants associated with Darier disease elicits ER stress and apoptosis in keratinocytes
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    Article Title: Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival
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    Article Title: Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)
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    Article Title: NPV-LDE-225 (Erismodegib) inhibits epithelial mesenchymal transition and self-renewal of glioblastoma initiating cells by regulating miR-21, miR-128, and miR-200
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    Centrifugation:

    Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation
    Article Snippet: .. On the contrary, to extract RNA from the clones obtained by transduction with lentiviral vector, cells were collected by centrifugation at 1.200 rpm for 5 min at 4 °C, washed with PBS, lysed with 1 ml of TRIZOL® Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer instructions. .. For gene expression analysis 1 μg of total RNA was reverse transcribed by using random hexamers.

    Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation
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    Amplification:

    Article Title: Protein aggregation of SERCA2 mutants associated with Darier disease elicits ER stress and apoptosis in keratinocytes
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    Polymerase Chain Reaction:

    Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation
    Article Snippet: PCR primers and probe were: A γ globin forward primer, 5′-TGG CAA GAA GGT GCT GAC TTC-3′, A γ globin reverse primer, 5’-TCA CTC AGC TGG GCA AAG G-3′, A γ globin probe, 5’-FAM-TGG GAG ATG CCA TAA AGC ACC TGG-TAMRA-3′. .. On the contrary, to extract RNA from the clones obtained by transduction with lentiviral vector, cells were collected by centrifugation at 1.200 rpm for 5 min at 4 °C, washed with PBS, lysed with 1 ml of TRIZOL® Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer instructions.

    Article Title: Protein aggregation of SERCA2 mutants associated with Darier disease elicits ER stress and apoptosis in keratinocytes
    Article Snippet: The coding region of mouse Serca2b cDNA was amplified by PCR using reverse transcripts of freshly isolated C57BL/6J mouse brain mRNA as template, and cloned into a pcDNA vector with a FLAG tag at the N-terminus of Serca2b, named SERCA2 in the text. .. SERCA2 , G23E and C268F were subcloned from their pcDNA plasmids into a lentiviral vector of pLenti6/V5-TOPO (Invitrogen) after the lentiviral vector was modified.

    Article Title: Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)
    Article Snippet: Using the cDNA, reverse transcription PCR (RT-PCR) was performed to amplify the coding sequence of the SV40 large T antigen (SV40_LTEG_exp_F; 5′-GGC GCC ATG GAT AAA GTT TTA AAC AGA GAG GA-3′, SV40_LTEG_exp_R; 5′-TTA TGT TTC AGG TTC AGG GGG AG-3′) and hTERT (hTERT_exp_F; 5′-ACC CCC GCG ATG CCG CGC GCT CCC-3′, hTERT_exp_R; 5′-GGG TGA GGT GAG GTG TCA CCA ACA AG-3′) genes. .. The amplified products were cloned into a commercial lentiviral vector (pLenti6.3/V5-TOPO; Life Technologies) and were validated by sequencing analysis using a primer pair (CMV_seq_F; 5′-CGC AAA TGG GCG GTA GGC GTG-3′, V5_seq_R; 5′-ACC GAG GAG AGG GTT AGG GAT-3′).

    Construct:

    Article Title: Tumor-Targeted Immunotherapy by Using Primary Adipose-Derived Stem Cells and an Antigen-Specific Protein Vaccine
    Article Snippet: .. The lentiviral vector (pLL3.7-E7’-eGFP) and the packaging constructs (pMDL g/p RRE, p RSV-REV, and pMD2.G) were transfected into 293FT cells (Invitrogen, Fisher Scientific, Carlsbad, CA, USA) with Lipofectamine 2000 Transfection Reagent (Invitrogen). .. Infectious viral particles were collected 48 h after transfection.

    Article Title: Characterization of cytoplasmic cyclin D1 as a marker of invasiveness in cancer
    Article Snippet: Expression vectors Human CCND1 wild type and CCND1K112E inactive allele were used to obtain an N-terminal 3xHA fusion under the UBI promoter in a lentiviral vector derived from pDSL (Invitrogen) or under the CMV promoter in a pCDNA3 vector. .. In these constructs the 3′ end of the K-Ras ORF containing the CAAX motif (GGC TGT GTG AAA ATT AAA AAA TGC ATT ATA ATG TAA) was fused to the 3′ end of the CCND1 ORF.

    Article Title: Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice
    Article Snippet: The human SGSH cDNA sequence including 5′ and 3′ UTR flanked by attB sites from image clone #5226903 (Geneservice, Paisley, UK) was recombined in a Gateway cloning reaction with BP Clonase into a donor vector (pDonor221) containing attP sites and then into the lentiviral vector using LR Clonase to create pHRsin.SFFV.hSGSH.att.wpre as described (Invitrogen Life Technologies). .. To confirm the ability of microglia to produce active enzyme from the lentiviral construct, the human microglial cell line CHME3 was transduced at an multiplicity of infection of 10 with lentiviral vector.

    Real-time Polymerase Chain Reaction:

    Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation
    Article Snippet: Quantitative real-time PCR assay was carried out using two different instruments: iQ™5 Multicolor Real-Time PCR Detection System and CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratoires, Hercules, CA, USA). .. On the contrary, to extract RNA from the clones obtained by transduction with lentiviral vector, cells were collected by centrifugation at 1.200 rpm for 5 min at 4 °C, washed with PBS, lysed with 1 ml of TRIZOL® Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer instructions.

    Incubation:

    Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation
    Article Snippet: For the in vivo experiments, embryonic mesoangioblasts (D16) were transduced with a lentiviral vector encoding for nuclear LacZ, whereas adult mesoangioblasts (G1) were labeled with DiI (Molecular Probes, Inc.). .. In brief, 2 × 107 bone marrow cells, obtained by flushing femurs and tibias with PBS containing 2% FCS, were subjected to centrifugation and were resuspended in 1 ml of the same flushing buffer; 10 μl SpinSep™ antibody cocktail (containing antibodies against CD5, CD45R, CD11b, TER 119, Gr-1, and neutrophils) was added to the cell suspension and incubated at 4°C for 30 min. After washing, 100 μl of the SpinSep™-dense particles was added to the cells and incubated on ice for 20 min.

    Activity Assay:

    Article Title: Characterization of cytoplasmic cyclin D1 as a marker of invasiveness in cancer
    Article Snippet: Expression vectors Human CCND1 wild type and CCND1K112E inactive allele were used to obtain an N-terminal 3xHA fusion under the UBI promoter in a lentiviral vector derived from pDSL (Invitrogen) or under the CMV promoter in a pCDNA3 vector. .. For RalB activity assay we performed an N-terminal 3xHA fusion to human RalB (image 3880116) and cloned in a pCDNA3 vector.

    Article Title: Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice
    Article Snippet: The human SGSH cDNA sequence including 5′ and 3′ UTR flanked by attB sites from image clone #5226903 (Geneservice, Paisley, UK) was recombined in a Gateway cloning reaction with BP Clonase into a donor vector (pDonor221) containing attP sites and then into the lentiviral vector using LR Clonase to create pHRsin.SFFV.hSGSH.att.wpre as described (Invitrogen Life Technologies). .. Twenty four hours later the media was changed and 48 hours after that the media and cells were collected for analysis of SGSH activity.

    Article Title: An RNA editing fingerprint of cancer stem cell reprogramming
    Article Snippet: Lentiviral vector preparation and ADAR1 site-directed mutagenesis We have previously characterized lentiviral vectors (Thermo Scientific) for overexpression of human ADAR1 p150-IRES-GFP [ ]. .. Mutagenic primers were designed to produce a nucleotide substitution of A5293C, which generates an E912A amino acid change and abolishes RNA editase activity [ ].

    Cell Culture:

    Article Title: Tumor-Targeted Immunotherapy by Using Primary Adipose-Derived Stem Cells and an Antigen-Specific Protein Vaccine
    Article Snippet: ADSCs were isolated and cultured as previously described [ ]. .. The lentiviral vector (pLL3.7-E7’-eGFP) and the packaging constructs (pMDL g/p RRE, p RSV-REV, and pMD2.G) were transfected into 293FT cells (Invitrogen, Fisher Scientific, Carlsbad, CA, USA) with Lipofectamine 2000 Transfection Reagent (Invitrogen).

    Expressing:

    Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation
    Article Snippet: On the contrary, to extract RNA from the clones obtained by transduction with lentiviral vector, cells were collected by centrifugation at 1.200 rpm for 5 min at 4 °C, washed with PBS, lysed with 1 ml of TRIZOL® Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer instructions. .. For gene expression analysis 1 μg of total RNA was reverse transcribed by using random hexamers.

    Article Title: NVP-LDE-225 (Erismodegib) inhibits epithelial-mesenchymal transition and human prostate cancer stem cell growth in NOD/SCID IL2R? null mice by regulating Bmi-1 and microRNA-128
    Article Snippet: In brief, 293T cells were transfected with 4 μg of plasmid and 4 μg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Life Technologies) according to the manufacturer's protocol. .. Prostate CSCs were transduced with lentivirus expressing scrambled (control) or shRNA against specific genes.

    Article Title: Characterization of cytoplasmic cyclin D1 as a marker of invasiveness in cancer
    Article Snippet: .. Expression vectors Human CCND1 wild type and CCND1K112E inactive allele were used to obtain an N-terminal 3xHA fusion under the UBI promoter in a lentiviral vector derived from pDSL (Invitrogen) or under the CMV promoter in a pCDNA3 vector. .. In these constructs the 3′ end of the K-Ras ORF containing the CAAX motif (GGC TGT GTG AAA ATT AAA AAA TGC ATT ATA ATG TAA) was fused to the 3′ end of the CCND1 ORF.

    Article Title: NPV-LDE-225 (Erismodegib) inhibits epithelial mesenchymal transition and self-renewal of glioblastoma initiating cells by regulating miR-21, miR-128, and miR-200
    Article Snippet: In brief, 293T cells were transfected with 4 µg of plasmid and 4 µg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Invitrogen) according to the manufacturer's protocol. .. Brain GICs were transduced with lentivirus expressing scrambled shRNA (control), Gli1 shRNA, or Gli2 shRNA.

    Article Title: Epstein-Barr virus nuclear antigen 5 inhibits pre-mRNA cleavage and polyadenylation
    Article Snippet: The resulting plasmid, pCI-EBNA5, contains the EBNA5 reading frame under the control of the CMV promoter. pcDNA6-EBNA5 is an expression vector for EBNA5. .. The EBNA5-encoding region was excised from the pCI-EBNA5 plasmid with Nhe I and Eco RI and ligated into Nhe I + Eco RI-opened pcDNA6/V5-His A vector (Invitrogen).

    Modification:

    Article Title: Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice
    Article Snippet: The lentiviral vector plasmid pHR'SIN-cPPT-SEW was modified by replacing the eGFP gene with a Gateway conversion cassette (Invitrogen Life Technologies, Paisley, UK # 11828-019) producing pHRsin.SFFV.Gateway. .. The human SGSH cDNA sequence including 5′ and 3′ UTR flanked by attB sites from image clone #5226903 (Geneservice, Paisley, UK) was recombined in a Gateway cloning reaction with BP Clonase into a donor vector (pDonor221) containing attP sites and then into the lentiviral vector using LR Clonase to create pHRsin.SFFV.hSGSH.att.wpre as described (Invitrogen Life Technologies).

    Article Title: Protein aggregation of SERCA2 mutants associated with Darier disease elicits ER stress and apoptosis in keratinocytes
    Article Snippet: .. SERCA2 , G23E and C268F were subcloned from their pcDNA plasmids into a lentiviral vector of pLenti6/V5-TOPO (Invitrogen) after the lentiviral vector was modified. .. The V5 tag was fused into the C-terminal sequence of SERCA2 or its mutants.

    Article Title: NPV-LDE-225 (Erismodegib) inhibits epithelial mesenchymal transition and self-renewal of glioblastoma initiating cells by regulating miR-21, miR-128, and miR-200
    Article Snippet: Packaging 293T cells were plated in 10-cm plates at a cell density of 5 × 106 a day prior to transfection in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum without antibiotics. .. In brief, 293T cells were transfected with 4 µg of plasmid and 4 µg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Invitrogen) according to the manufacturer's protocol.

    Over Expression:

    Article Title: An RNA editing fingerprint of cancer stem cell reprogramming
    Article Snippet: .. Lentiviral vector preparation and ADAR1 site-directed mutagenesis We have previously characterized lentiviral vectors (Thermo Scientific) for overexpression of human ADAR1 p150-IRES-GFP [ ]. .. For production of the catalytically-inactive ADAR1 mutated (ADAR1m) lentiviral vector, site-directed mutagenesis was carried out using the QuikChange II Site-Directed Mutagenesis Kit (Agilent) according to manufacturer’s instructions.

    Derivative Assay:

    Article Title: Characterization of cytoplasmic cyclin D1 as a marker of invasiveness in cancer
    Article Snippet: .. Expression vectors Human CCND1 wild type and CCND1K112E inactive allele were used to obtain an N-terminal 3xHA fusion under the UBI promoter in a lentiviral vector derived from pDSL (Invitrogen) or under the CMV promoter in a pCDNA3 vector. .. In these constructs the 3′ end of the K-Ras ORF containing the CAAX motif (GGC TGT GTG AAA ATT AAA AAA TGC ATT ATA ATG TAA) was fused to the 3′ end of the CCND1 ORF.

    Article Title: Epstein-Barr virus nuclear antigen 5 inhibits pre-mRNA cleavage and polyadenylation
    Article Snippet: The EBNA5-encoding reading frame in this plasmid is derived from the IB4 cell line and contains seven copies of the W1 and W2 exons and one copy each of the Y1 and Y2 exons. .. The EBNA5-encoding region was excised from the pCI-EBNA5 plasmid with Nhe I and Eco RI and ligated into Nhe I + Eco RI-opened pcDNA6/V5-His A vector (Invitrogen).

    Countercurrent Chromatography:

    Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation
    Article Snippet: On the contrary, to extract RNA from the clones obtained by transduction with lentiviral vector, cells were collected by centrifugation at 1.200 rpm for 5 min at 4 °C, washed with PBS, lysed with 1 ml of TRIZOL® Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer instructions. .. For quantitative real-time PCR analysis of UPF1 mRNA, we used the human UPF1 expression assay kit (Thermo Fisher Scientific, Waltham, MA, USA); while for real-time PCR analysis of β globin mRNA, the following primers were used: β globin forward primer, 5’-CAA GAA AGT GCT CGG TGC CT-3′; β globin reverse primer, 5’-GCA AAG GTG CCC TTG AGG T-3′; βglobin probe, 5’-FAM-TAG TGA TGG CCT GGC TCA CCT GGA C-MGB-3′.

    Article Title: Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)
    Article Snippet: Using the cDNA, reverse transcription PCR (RT-PCR) was performed to amplify the coding sequence of the SV40 large T antigen (SV40_LTEG_exp_F; 5′-GGC GCC ATG GAT AAA GTT TTA AAC AGA GAG GA-3′, SV40_LTEG_exp_R; 5′-TTA TGT TTC AGG TTC AGG GGG AG-3′) and hTERT (hTERT_exp_F; 5′-ACC CCC GCG ATG CCG CGC GCT CCC-3′, hTERT_exp_R; 5′-GGG TGA GGT GAG GTG TCA CCA ACA AG-3′) genes. .. The amplified products were cloned into a commercial lentiviral vector (pLenti6.3/V5-TOPO; Life Technologies) and were validated by sequencing analysis using a primer pair (CMV_seq_F; 5′-CGC AAA TGG GCG GTA GGC GTG-3′, V5_seq_R; 5′-ACC GAG GAG AGG GTT AGG GAT-3′).

    Transfection:

    Article Title: Tumor-Targeted Immunotherapy by Using Primary Adipose-Derived Stem Cells and an Antigen-Specific Protein Vaccine
    Article Snippet: .. The lentiviral vector (pLL3.7-E7’-eGFP) and the packaging constructs (pMDL g/p RRE, p RSV-REV, and pMD2.G) were transfected into 293FT cells (Invitrogen, Fisher Scientific, Carlsbad, CA, USA) with Lipofectamine 2000 Transfection Reagent (Invitrogen). .. Infectious viral particles were collected 48 h after transfection.

    Article Title: NVP-LDE-225 (Erismodegib) inhibits epithelial-mesenchymal transition and human prostate cancer stem cell growth in NOD/SCID IL2R? null mice by regulating Bmi-1 and microRNA-128
    Article Snippet: .. In brief, 293T cells were transfected with 4 μg of plasmid and 4 μg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Life Technologies) according to the manufacturer's protocol. .. Viral supernatants were collected and concentrated by adding PEG-it virus precipitation solution (SBI System Biosciences) to produce virus stocks with titers of 1 × 108 –1 × 109 infectious units/ml.

    Article Title: Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)
    Article Snippet: The amplified products were cloned into a commercial lentiviral vector (pLenti6.3/V5-TOPO; Life Technologies) and were validated by sequencing analysis using a primer pair (CMV_seq_F; 5′-CGC AAA TGG GCG GTA GGC GTG-3′, V5_seq_R; 5′-ACC GAG GAG AGG GTT AGG GAT-3′). .. The lentiviral vectors were transfected to 293T cells along with a mixture of 3 packaging plasmids (ViraPower Packaging Mix; Life Technologies Corporation) using a commercial transfection reagent (FuGENE HD, Promega Corporation, Madison, Wisconsin).

    Article Title: NPV-LDE-225 (Erismodegib) inhibits epithelial mesenchymal transition and self-renewal of glioblastoma initiating cells by regulating miR-21, miR-128, and miR-200
    Article Snippet: .. In brief, 293T cells were transfected with 4 µg of plasmid and 4 µg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Invitrogen) according to the manufacturer's protocol. .. Viral supernatants were collected and concentrated by adding PEG-it virus precipitation solution (SBI System Biosciences) to produce virus stocks with titers of 1 × 108 to 1 × 109 infectious units per milliliter.

    Infection:

    Article Title: Tumor-Targeted Immunotherapy by Using Primary Adipose-Derived Stem Cells and an Antigen-Specific Protein Vaccine
    Article Snippet: The lentiviral vector (pLL3.7-E7’-eGFP) and the packaging constructs (pMDL g/p RRE, p RSV-REV, and pMD2.G) were transfected into 293FT cells (Invitrogen, Fisher Scientific, Carlsbad, CA, USA) with Lipofectamine 2000 Transfection Reagent (Invitrogen). .. ADSCs were infected with appropriate virus titers in media containing 8 μg/mL polybrene.

    Article Title: NVP-LDE-225 (Erismodegib) inhibits epithelial-mesenchymal transition and human prostate cancer stem cell growth in NOD/SCID IL2R? null mice by regulating Bmi-1 and microRNA-128
    Article Snippet: Transfection of packaging cells and infection of prostate CSCs were carried out using standard protocols with some modifications. .. In brief, 293T cells were transfected with 4 μg of plasmid and 4 μg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Life Technologies) according to the manufacturer's protocol.

    Article Title: Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice
    Article Snippet: The human SGSH cDNA sequence including 5′ and 3′ UTR flanked by attB sites from image clone #5226903 (Geneservice, Paisley, UK) was recombined in a Gateway cloning reaction with BP Clonase into a donor vector (pDonor221) containing attP sites and then into the lentiviral vector using LR Clonase to create pHRsin.SFFV.hSGSH.att.wpre as described (Invitrogen Life Technologies). .. To confirm the ability of microglia to produce active enzyme from the lentiviral construct, the human microglial cell line CHME3 was transduced at an multiplicity of infection of 10 with lentiviral vector.

    Article Title: Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival
    Article Snippet: A set of human RNH1 -specific shRNA cloned in pLKO.1 lentiviral vector was purchased from Open Biosystems. .. HeLa cells were infected with lentivirus in the presence of 10 µg/ml polybrene for 48 hours.

    Article Title: NPV-LDE-225 (Erismodegib) inhibits epithelial mesenchymal transition and self-renewal of glioblastoma initiating cells by regulating miR-21, miR-128, and miR-200
    Article Snippet: Transfection of packaging cells and infection of brain GICs were carried out using standard protocols with some modifications. .. In brief, 293T cells were transfected with 4 µg of plasmid and 4 µg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Invitrogen) according to the manufacturer's protocol.

    Sequencing:

    Article Title: Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice
    Article Snippet: .. The human SGSH cDNA sequence including 5′ and 3′ UTR flanked by attB sites from image clone #5226903 (Geneservice, Paisley, UK) was recombined in a Gateway cloning reaction with BP Clonase into a donor vector (pDonor221) containing attP sites and then into the lentiviral vector using LR Clonase to create pHRsin.SFFV.hSGSH.att.wpre as described (Invitrogen Life Technologies). .. To confirm the ability of microglia to produce active enzyme from the lentiviral construct, the human microglial cell line CHME3 was transduced at an multiplicity of infection of 10 with lentiviral vector.

    Article Title: Protein aggregation of SERCA2 mutants associated with Darier disease elicits ER stress and apoptosis in keratinocytes
    Article Snippet: After sequencing confirmation of the wild-type Serca2b , all mutants studied were made by site-directed mutagenesis using Serca2b as a template, and confirmed by sequencing again. .. SERCA2 , G23E and C268F were subcloned from their pcDNA plasmids into a lentiviral vector of pLenti6/V5-TOPO (Invitrogen) after the lentiviral vector was modified.

    Article Title: Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)
    Article Snippet: .. The amplified products were cloned into a commercial lentiviral vector (pLenti6.3/V5-TOPO; Life Technologies) and were validated by sequencing analysis using a primer pair (CMV_seq_F; 5′-CGC AAA TGG GCG GTA GGC GTG-3′, V5_seq_R; 5′-ACC GAG GAG AGG GTT AGG GAT-3′). .. The lentiviral vectors were transfected to 293T cells along with a mixture of 3 packaging plasmids (ViraPower Packaging Mix; Life Technologies Corporation) using a commercial transfection reagent (FuGENE HD, Promega Corporation, Madison, Wisconsin).

    Antiviral Assay:

    Article Title: Epstein-Barr virus nuclear antigen 5 inhibits pre-mRNA cleavage and polyadenylation
    Article Snippet: The EBNA5-encoding region was excised from the pCI-EBNA5 plasmid with Nhe I and Eco RI and ligated into Nhe I + Eco RI-opened pcDNA6/V5-His A vector (Invitrogen). .. The Ava I– Ava I Wp fragment was excised from pBΔWCAT-1 and cloned in pCATGem using Hin dIII linkers.

    Cellular Antioxidant Activity Assay:

    Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation
    Article Snippet: PCR primers and probe were: A γ globin forward primer, 5′-TGG CAA GAA GGT GCT GAC TTC-3′, A γ globin reverse primer, 5’-TCA CTC AGC TGG GCA AAG G-3′, A γ globin probe, 5’-FAM-TGG GAG ATG CCA TAA AGC ACC TGG-TAMRA-3′. .. On the contrary, to extract RNA from the clones obtained by transduction with lentiviral vector, cells were collected by centrifugation at 1.200 rpm for 5 min at 4 °C, washed with PBS, lysed with 1 ml of TRIZOL® Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer instructions.

    In Vivo:

    Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation
    Article Snippet: .. For the in vivo experiments, embryonic mesoangioblasts (D16) were transduced with a lentiviral vector encoding for nuclear LacZ, whereas adult mesoangioblasts (G1) were labeled with DiI (Molecular Probes, Inc.). .. Lin− cells were isolated from CD-1 mice using the SpinSep™ kit (StemCell Technologies Inc.) according to the manufacturer's instructions.

    Mutagenesis:

    Article Title: Protein aggregation of SERCA2 mutants associated with Darier disease elicits ER stress and apoptosis in keratinocytes
    Article Snippet: After sequencing confirmation of the wild-type Serca2b , all mutants studied were made by site-directed mutagenesis using Serca2b as a template, and confirmed by sequencing again. .. SERCA2 , G23E and C268F were subcloned from their pcDNA plasmids into a lentiviral vector of pLenti6/V5-TOPO (Invitrogen) after the lentiviral vector was modified.

    Article Title: An RNA editing fingerprint of cancer stem cell reprogramming
    Article Snippet: .. Lentiviral vector preparation and ADAR1 site-directed mutagenesis We have previously characterized lentiviral vectors (Thermo Scientific) for overexpression of human ADAR1 p150-IRES-GFP [ ]. .. For production of the catalytically-inactive ADAR1 mutated (ADAR1m) lentiviral vector, site-directed mutagenesis was carried out using the QuikChange II Site-Directed Mutagenesis Kit (Agilent) according to manufacturer’s instructions.

    Isolation:

    Article Title: Tumor-Targeted Immunotherapy by Using Primary Adipose-Derived Stem Cells and an Antigen-Specific Protein Vaccine
    Article Snippet: Paragraph title: 4.2. Isolation, Culture, and Lentiviral Transduction of ADSCs ... The lentiviral vector (pLL3.7-E7’-eGFP) and the packaging constructs (pMDL g/p RRE, p RSV-REV, and pMD2.G) were transfected into 293FT cells (Invitrogen, Fisher Scientific, Carlsbad, CA, USA) with Lipofectamine 2000 Transfection Reagent (Invitrogen).

    Article Title: Protein aggregation of SERCA2 mutants associated with Darier disease elicits ER stress and apoptosis in keratinocytes
    Article Snippet: The coding region of mouse Serca2b cDNA was amplified by PCR using reverse transcripts of freshly isolated C57BL/6J mouse brain mRNA as template, and cloned into a pcDNA vector with a FLAG tag at the N-terminus of Serca2b, named SERCA2 in the text. .. SERCA2 , G23E and C268F were subcloned from their pcDNA plasmids into a lentiviral vector of pLenti6/V5-TOPO (Invitrogen) after the lentiviral vector was modified.

    Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation
    Article Snippet: Mesoangioblast cell lines were isolated from the dorsal aorta of mouse embryos (D16) or bone marrow (G1), cloned, and expanded as described previously ( ). .. For the in vivo experiments, embryonic mesoangioblasts (D16) were transduced with a lentiviral vector encoding for nuclear LacZ, whereas adult mesoangioblasts (G1) were labeled with DiI (Molecular Probes, Inc.).

    Labeling:

    Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation
    Article Snippet: .. For the in vivo experiments, embryonic mesoangioblasts (D16) were transduced with a lentiviral vector encoding for nuclear LacZ, whereas adult mesoangioblasts (G1) were labeled with DiI (Molecular Probes, Inc.). .. Lin− cells were isolated from CD-1 mice using the SpinSep™ kit (StemCell Technologies Inc.) according to the manufacturer's instructions.

    Mouse Assay:

    Article Title: Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice
    Article Snippet: The human SGSH cDNA sequence including 5′ and 3′ UTR flanked by attB sites from image clone #5226903 (Geneservice, Paisley, UK) was recombined in a Gateway cloning reaction with BP Clonase into a donor vector (pDonor221) containing attP sites and then into the lentiviral vector using LR Clonase to create pHRsin.SFFV.hSGSH.att.wpre as described (Invitrogen Life Technologies). .. Total bone marrow mononuclear cells from WT and MPS IIIA mice were lineage depleted using the murine hematopoietic progenitor enrichment cocktail (Stem Cell Technologies, Grenoble, France) according to manufacturer's instructions.

    Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation
    Article Snippet: For the in vivo experiments, embryonic mesoangioblasts (D16) were transduced with a lentiviral vector encoding for nuclear LacZ, whereas adult mesoangioblasts (G1) were labeled with DiI (Molecular Probes, Inc.). .. Lin− cells were isolated from CD-1 mice using the SpinSep™ kit (StemCell Technologies Inc.) according to the manufacturer's instructions.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Protein aggregation of SERCA2 mutants associated with Darier disease elicits ER stress and apoptosis in keratinocytes
    Article Snippet: Paragraph title: Plasmids and RT-PCR ... SERCA2 , G23E and C268F were subcloned from their pcDNA plasmids into a lentiviral vector of pLenti6/V5-TOPO (Invitrogen) after the lentiviral vector was modified.

    Article Title: Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)
    Article Snippet: Using the cDNA, reverse transcription PCR (RT-PCR) was performed to amplify the coding sequence of the SV40 large T antigen (SV40_LTEG_exp_F; 5′-GGC GCC ATG GAT AAA GTT TTA AAC AGA GAG GA-3′, SV40_LTEG_exp_R; 5′-TTA TGT TTC AGG TTC AGG GGG AG-3′) and hTERT (hTERT_exp_F; 5′-ACC CCC GCG ATG CCG CGC GCT CCC-3′, hTERT_exp_R; 5′-GGG TGA GGT GAG GTG TCA CCA ACA AG-3′) genes. .. The amplified products were cloned into a commercial lentiviral vector (pLenti6.3/V5-TOPO; Life Technologies) and were validated by sequencing analysis using a primer pair (CMV_seq_F; 5′-CGC AAA TGG GCG GTA GGC GTG-3′, V5_seq_R; 5′-ACC GAG GAG AGG GTT AGG GAT-3′).

    Plasmid Preparation:

    Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation
    Article Snippet: .. On the contrary, to extract RNA from the clones obtained by transduction with lentiviral vector, cells were collected by centrifugation at 1.200 rpm for 5 min at 4 °C, washed with PBS, lysed with 1 ml of TRIZOL® Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer instructions. .. For gene expression analysis 1 μg of total RNA was reverse transcribed by using random hexamers.

    Article Title: Tumor-Targeted Immunotherapy by Using Primary Adipose-Derived Stem Cells and an Antigen-Specific Protein Vaccine
    Article Snippet: .. The lentiviral vector (pLL3.7-E7’-eGFP) and the packaging constructs (pMDL g/p RRE, p RSV-REV, and pMD2.G) were transfected into 293FT cells (Invitrogen, Fisher Scientific, Carlsbad, CA, USA) with Lipofectamine 2000 Transfection Reagent (Invitrogen). .. Infectious viral particles were collected 48 h after transfection.

    Article Title: NVP-LDE-225 (Erismodegib) inhibits epithelial-mesenchymal transition and human prostate cancer stem cell growth in NOD/SCID IL2R? null mice by regulating Bmi-1 and microRNA-128
    Article Snippet: .. In brief, 293T cells were transfected with 4 μg of plasmid and 4 μg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Life Technologies) according to the manufacturer's protocol. .. Viral supernatants were collected and concentrated by adding PEG-it virus precipitation solution (SBI System Biosciences) to produce virus stocks with titers of 1 × 108 –1 × 109 infectious units/ml.

    Article Title: Characterization of cytoplasmic cyclin D1 as a marker of invasiveness in cancer
    Article Snippet: .. Expression vectors Human CCND1 wild type and CCND1K112E inactive allele were used to obtain an N-terminal 3xHA fusion under the UBI promoter in a lentiviral vector derived from pDSL (Invitrogen) or under the CMV promoter in a pCDNA3 vector. .. In these constructs the 3′ end of the K-Ras ORF containing the CAAX motif (GGC TGT GTG AAA ATT AAA AAA TGC ATT ATA ATG TAA) was fused to the 3′ end of the CCND1 ORF.

    Article Title: Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice
    Article Snippet: .. The human SGSH cDNA sequence including 5′ and 3′ UTR flanked by attB sites from image clone #5226903 (Geneservice, Paisley, UK) was recombined in a Gateway cloning reaction with BP Clonase into a donor vector (pDonor221) containing attP sites and then into the lentiviral vector using LR Clonase to create pHRsin.SFFV.hSGSH.att.wpre as described (Invitrogen Life Technologies). .. To confirm the ability of microglia to produce active enzyme from the lentiviral construct, the human microglial cell line CHME3 was transduced at an multiplicity of infection of 10 with lentiviral vector.

    Article Title: Protein aggregation of SERCA2 mutants associated with Darier disease elicits ER stress and apoptosis in keratinocytes
    Article Snippet: .. SERCA2 , G23E and C268F were subcloned from their pcDNA plasmids into a lentiviral vector of pLenti6/V5-TOPO (Invitrogen) after the lentiviral vector was modified. .. The V5 tag was fused into the C-terminal sequence of SERCA2 or its mutants.

    Article Title: An RNA editing fingerprint of cancer stem cell reprogramming
    Article Snippet: .. Lentiviral vector preparation and ADAR1 site-directed mutagenesis We have previously characterized lentiviral vectors (Thermo Scientific) for overexpression of human ADAR1 p150-IRES-GFP [ ]. .. For production of the catalytically-inactive ADAR1 mutated (ADAR1m) lentiviral vector, site-directed mutagenesis was carried out using the QuikChange II Site-Directed Mutagenesis Kit (Agilent) according to manufacturer’s instructions.

    Article Title: Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation
    Article Snippet: .. For the in vivo experiments, embryonic mesoangioblasts (D16) were transduced with a lentiviral vector encoding for nuclear LacZ, whereas adult mesoangioblasts (G1) were labeled with DiI (Molecular Probes, Inc.). .. Lin− cells were isolated from CD-1 mice using the SpinSep™ kit (StemCell Technologies Inc.) according to the manufacturer's instructions.

    Article Title: Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival
    Article Snippet: .. A set of human RNH1 -specific shRNA cloned in pLKO.1 lentiviral vector was purchased from Open Biosystems. .. Lentiviral particles were packaged in HEK293T cells co-transfected with shRNA inserted pLKO.1 (5.8 µg), envelope plasmid pMD2.G (1.8 µg) and packaging plasmid psPAX (4.4 µg).

    Article Title: Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)
    Article Snippet: .. The amplified products were cloned into a commercial lentiviral vector (pLenti6.3/V5-TOPO; Life Technologies) and were validated by sequencing analysis using a primer pair (CMV_seq_F; 5′-CGC AAA TGG GCG GTA GGC GTG-3′, V5_seq_R; 5′-ACC GAG GAG AGG GTT AGG GAT-3′). .. The lentiviral vectors were transfected to 293T cells along with a mixture of 3 packaging plasmids (ViraPower Packaging Mix; Life Technologies Corporation) using a commercial transfection reagent (FuGENE HD, Promega Corporation, Madison, Wisconsin).

    Article Title: NPV-LDE-225 (Erismodegib) inhibits epithelial mesenchymal transition and self-renewal of glioblastoma initiating cells by regulating miR-21, miR-128, and miR-200
    Article Snippet: .. In brief, 293T cells were transfected with 4 µg of plasmid and 4 µg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Invitrogen) according to the manufacturer's protocol. .. Viral supernatants were collected and concentrated by adding PEG-it virus precipitation solution (SBI System Biosciences) to produce virus stocks with titers of 1 × 108 to 1 × 109 infectious units per milliliter.

    Article Title: Epstein-Barr virus nuclear antigen 5 inhibits pre-mRNA cleavage and polyadenylation
    Article Snippet: .. The EBNA5-encoding region was excised from the pCI-EBNA5 plasmid with Nhe I and Eco RI and ligated into Nhe I + Eco RI-opened pcDNA6/V5-His A vector (Invitrogen). .. The EBNA2 expression vector pEΔA6 has been described previously ( ).

    shRNA:

    Article Title: UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation
    Article Snippet: Paragraph title: Production and characterization of K562-derived cell clones by UPF1 shRNA lentivirus vectors ... On the contrary, to extract RNA from the clones obtained by transduction with lentiviral vector, cells were collected by centrifugation at 1.200 rpm for 5 min at 4 °C, washed with PBS, lysed with 1 ml of TRIZOL® Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer instructions.

    Article Title: NVP-LDE-225 (Erismodegib) inhibits epithelial-mesenchymal transition and human prostate cancer stem cell growth in NOD/SCID IL2R? null mice by regulating Bmi-1 and microRNA-128
    Article Snippet: In brief, 293T cells were transfected with 4 μg of plasmid and 4 μg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Life Technologies) according to the manufacturer's protocol. .. Prostate CSCs were transduced with lentivirus expressing scrambled (control) or shRNA against specific genes.

    Article Title: Characterization of cytoplasmic cyclin D1 as a marker of invasiveness in cancer
    Article Snippet: Expression vectors Human CCND1 wild type and CCND1K112E inactive allele were used to obtain an N-terminal 3xHA fusion under the UBI promoter in a lentiviral vector derived from pDSL (Invitrogen) or under the CMV promoter in a pCDNA3 vector. .. The RALB MISSION shRNA TRCN0000072957 and the CCND1 MISSION shRNA TRCN0000026883, both cloned in a pLKO.1-puro, were obtained from Sigma.

    Article Title: Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival
    Article Snippet: .. A set of human RNH1 -specific shRNA cloned in pLKO.1 lentiviral vector was purchased from Open Biosystems. .. Lentiviral particles were packaged in HEK293T cells co-transfected with shRNA inserted pLKO.1 (5.8 µg), envelope plasmid pMD2.G (1.8 µg) and packaging plasmid psPAX (4.4 µg).

    Article Title: NPV-LDE-225 (Erismodegib) inhibits epithelial mesenchymal transition and self-renewal of glioblastoma initiating cells by regulating miR-21, miR-128, and miR-200
    Article Snippet: Paragraph title: Lentiviral Particle Production and Gli1 and Gli2 shRNA Transduction ... In brief, 293T cells were transfected with 4 µg of plasmid and 4 µg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Invitrogen) according to the manufacturer's protocol.

    Produced:

    Article Title: NPV-LDE-225 (Erismodegib) inhibits epithelial mesenchymal transition and self-renewal of glioblastoma initiating cells by regulating miR-21, miR-128, and miR-200
    Article Snippet: Lentivirus was produced by triple transfection of human embryonic kidney 293T cells. .. In brief, 293T cells were transfected with 4 µg of plasmid and 4 µg of lentiviral vector using lipid transfection (Lipofectamine-2000/Plus reagent, Invitrogen) according to the manufacturer's protocol.

    FLAG-tag:

    Article Title: Protein aggregation of SERCA2 mutants associated with Darier disease elicits ER stress and apoptosis in keratinocytes
    Article Snippet: The coding region of mouse Serca2b cDNA was amplified by PCR using reverse transcripts of freshly isolated C57BL/6J mouse brain mRNA as template, and cloned into a pcDNA vector with a FLAG tag at the N-terminus of Serca2b, named SERCA2 in the text. .. SERCA2 , G23E and C268F were subcloned from their pcDNA plasmids into a lentiviral vector of pLenti6/V5-TOPO (Invitrogen) after the lentiviral vector was modified.

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    ptripz based doxycyline dox inducible lentiviral vectors - by Bioz Stars, 2020-04
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