Structured Review

Lonza gfp vector
<t>BATF</t> silencing enhanced cytokine secretions by PPD-specific T cells in PBMCs in patients with ATB. (A) Representative fluorescence microscopy image of transfected primary human PBMCs after 24 h of electroporation with 2 μg <t>GFP</t> Vector, and red arrow indicated transfected PBMCs containing GFP (× 200). (B) Transfection efficiency of primary human PBMCs 24 h post electroporation were assessed by analysis of GFP protein expression by flow cytometry. (C) Silencing of BATF by a BATF siRNA SMART pool in primary human PBMCs from ATB patients measured by real-time quantitative PCR. Expression normalized to a housekeeping gene ( GAPDH ) is presented as fold change relative to negative control siRNA. (D) PBMCs were electroporated with indicated siRNA, then cultured with M. tb -specific antigen PPD (25 μg/mL) overnight, and IL-2 or IFN-γ mRNA levels were measured by real-time quantitative PCR. The information of controls and cell viability after transfection was provided in the Methods section BATF Small Interfering RNA (siRNA) Knockdown in ATB Patients ( 11 ). Data are expressed as mean ± SEM ( n = 7). * P
Gfp Vector, supplied by Lonza, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "BATF Potentially Mediates Negative Regulation of PD-1/PD-Ls Pathway on T Cell Functions in Mycobacterium tuberculosis Infection"

Article Title: BATF Potentially Mediates Negative Regulation of PD-1/PD-Ls Pathway on T Cell Functions in Mycobacterium tuberculosis Infection

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.02430

BATF silencing enhanced cytokine secretions by PPD-specific T cells in PBMCs in patients with ATB. (A) Representative fluorescence microscopy image of transfected primary human PBMCs after 24 h of electroporation with 2 μg GFP Vector, and red arrow indicated transfected PBMCs containing GFP (× 200). (B) Transfection efficiency of primary human PBMCs 24 h post electroporation were assessed by analysis of GFP protein expression by flow cytometry. (C) Silencing of BATF by a BATF siRNA SMART pool in primary human PBMCs from ATB patients measured by real-time quantitative PCR. Expression normalized to a housekeeping gene ( GAPDH ) is presented as fold change relative to negative control siRNA. (D) PBMCs were electroporated with indicated siRNA, then cultured with M. tb -specific antigen PPD (25 μg/mL) overnight, and IL-2 or IFN-γ mRNA levels were measured by real-time quantitative PCR. The information of controls and cell viability after transfection was provided in the Methods section BATF Small Interfering RNA (siRNA) Knockdown in ATB Patients ( 11 ). Data are expressed as mean ± SEM ( n = 7). * P
Figure Legend Snippet: BATF silencing enhanced cytokine secretions by PPD-specific T cells in PBMCs in patients with ATB. (A) Representative fluorescence microscopy image of transfected primary human PBMCs after 24 h of electroporation with 2 μg GFP Vector, and red arrow indicated transfected PBMCs containing GFP (× 200). (B) Transfection efficiency of primary human PBMCs 24 h post electroporation were assessed by analysis of GFP protein expression by flow cytometry. (C) Silencing of BATF by a BATF siRNA SMART pool in primary human PBMCs from ATB patients measured by real-time quantitative PCR. Expression normalized to a housekeeping gene ( GAPDH ) is presented as fold change relative to negative control siRNA. (D) PBMCs were electroporated with indicated siRNA, then cultured with M. tb -specific antigen PPD (25 μg/mL) overnight, and IL-2 or IFN-γ mRNA levels were measured by real-time quantitative PCR. The information of controls and cell viability after transfection was provided in the Methods section BATF Small Interfering RNA (siRNA) Knockdown in ATB Patients ( 11 ). Data are expressed as mean ± SEM ( n = 7). * P

Techniques Used: Fluorescence, Microscopy, Transfection, Electroporation, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Negative Control, Cell Culture, Small Interfering RNA

2) Product Images from "Glia Maturation Factor-γ Regulates Monocyte Migration through Modulation of β1-Integrin *"

Article Title: Glia Maturation Factor-γ Regulates Monocyte Migration through Modulation of β1-Integrin *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.674200

GMFG overexpression enhances chemoattractant-stimulated cell migration and adhesion to FN. Human monocytes or THP-1 cells were transfected with GFP vector or GFP-tagged GMFG plasmid for 48 h. A , Western blotting analysis of expression of GMFG or GMFG-GFP
Figure Legend Snippet: GMFG overexpression enhances chemoattractant-stimulated cell migration and adhesion to FN. Human monocytes or THP-1 cells were transfected with GFP vector or GFP-tagged GMFG plasmid for 48 h. A , Western blotting analysis of expression of GMFG or GMFG-GFP

Techniques Used: Over Expression, Migration, Transfection, Plasmid Preparation, Western Blot, Expressing

Related Articles

Transfection:

Article Title: Characterization of cxorf21 Provides Molecular Insight Into Female-Bias Immune Response in SLE Pathogenesis
Article Snippet: .. To confirm transfection efficiency, control wells were simultaneously transfected with GFP vector (Lonza). .. The adherent monocyte cells were stained with DAPI and accessed under the microscope for GFP 24 h post-transfection.

Article Title: Glia Maturation Factor-γ Regulates Monocyte Migration through Modulation of β1-Integrin *
Article Snippet: .. Monocytes (1 × 106 cells) or THP-1 cells (2 × 106 cells) were transiently transfected with a GMFG siRNA, STX4 siRNA, STXBP4 siRNA, control negative siRNA, GFP vector, and/or GMFG-GFP vector using the Nucleofector Kit V and Nucleofector I Program U-01 (Lonza), according to the manufacturer's protocol. .. The GMFG siRNA (Silencer Select siRNA s18302), STXBP4 siRNA (Silencer Select siRNA s48386), STX4 siRNA (Silencer Select siRNA s13595), and control negative siRNA (Neg-siRNA-2) were purchased from Applied Biosystems.

Article Title: MMP-12, Secreted by Pro-Inflammatory Macrophages, Targets Endoglin in Human Macrophages and Endothelial Cells
Article Snippet: .. Control transfections with pDisplay empty vector (Invitrogen) or GFP vector (Lonza, Basel, Switzerland) were also included. .. Transfection experiments were performed with Lipofectamine LTX (Invitrogen) according to the manufacturer’s instructions.

Plasmid Preparation:

Article Title: Characterization of cxorf21 Provides Molecular Insight Into Female-Bias Immune Response in SLE Pathogenesis
Article Snippet: .. To confirm transfection efficiency, control wells were simultaneously transfected with GFP vector (Lonza). .. The adherent monocyte cells were stained with DAPI and accessed under the microscope for GFP 24 h post-transfection.

Article Title: Glia Maturation Factor-γ Regulates Monocyte Migration through Modulation of β1-Integrin *
Article Snippet: .. Monocytes (1 × 106 cells) or THP-1 cells (2 × 106 cells) were transiently transfected with a GMFG siRNA, STX4 siRNA, STXBP4 siRNA, control negative siRNA, GFP vector, and/or GMFG-GFP vector using the Nucleofector Kit V and Nucleofector I Program U-01 (Lonza), according to the manufacturer's protocol. .. The GMFG siRNA (Silencer Select siRNA s18302), STXBP4 siRNA (Silencer Select siRNA s48386), STX4 siRNA (Silencer Select siRNA s13595), and control negative siRNA (Neg-siRNA-2) were purchased from Applied Biosystems.

Article Title: A Systematic Strategy for Discovering a Therapeutic Drug for Alzheimer’s Disease and Its Target Molecule
Article Snippet: .. Briefly, mouse cortical neurons (5.0 × 106 cells) were mixed with siCRMP2 (1 μM, Thermo Scientific, cat # 4390771) or control siRNA (1 μM, Thermo Scientific, 4390843) and GFP vector (2 μg, Lonza, Basel, Switzerland) and electroporated with an Amaxa Nucleofector (Lonza). .. Three days after the transfection, cells were fixed and immunostained with a polyclonal antibody against CRMP2 (Sigma, cat # C2993).

Article Title: MMP-12, Secreted by Pro-Inflammatory Macrophages, Targets Endoglin in Human Macrophages and Endothelial Cells
Article Snippet: .. Control transfections with pDisplay empty vector (Invitrogen) or GFP vector (Lonza, Basel, Switzerland) were also included. .. Transfection experiments were performed with Lipofectamine LTX (Invitrogen) according to the manufacturer’s instructions.

Article Title: BATF Potentially Mediates Negative Regulation of PD-1/PD-Ls Pathway on T Cell Functions in Mycobacterium tuberculosis Infection
Article Snippet: .. Per 5 × 106 PBMCs from ATB patients were resuspended in 100 μL room-temperature balanced Nucleofector®solution (Human T Cell Nucleofector®Kit, Lonza, Germany), and added with 200 nM siRNA (ON-TARGET plus Non-targeting pool or BATF ON-TARGET plus SMART pool, GE Dharmacon, USA) or 2 μg GFP Vector (Lonza, Germany). .. Then, cells were electroporated according to manufacturer's introduction and incubated in 1mL pre-warmed Opti-MEM medium (Invitrogen-Gibco, USA) in a 12-well plate in 5% CO2 at 37°C.

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    Lonza plasmid cag human cd63 gfp
    Human <t>CD63-GFP</t> localization and extracellular vesicle (EV) analysis in the primary fibroblast cells obtained from the caudal vertebrae of Tg rats. ( a ) Localization of human CD63-GFP in the cultured Tg rat cells. Immunostaining indicated the co-localization of GFP with human CD63 (upper panels) and with rat CD63-positive signals (lower panels) around nuclei (blue). Scale bars = 50 μm. ( b ) Size distribution of the EVs isolated from the conditioned medium of Wt and Tg rat cells was determined using a NanoSight system. ( c , d ) The relationship between ceramide and the secretion of EVs. The intracellular rat CD63-positive signals and GFP signals were increased after treatment with 10 μM GW4869, a neutral sphingomyelinase (nSMase) inhibitor, for 24 hours ( c ). Western blotting showed a GW4869-dependent decrease of EV markers (rat CD63 and flotillin-1) and human CD63-GFP in the isolated EVs ( d ; left). However, the expression levels of rat CD63 and human CD63-GFP in the cell lysates were not changed by GW4869 ( d ; right). β-actin was used as a loading control. Both generation and protein composition of the EVs did not show an apparent change by CD63-GFP overexpression ( Supplementary Fig. 5 ).
    Plasmid Cag Human Cd63 Gfp, supplied by Lonza, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid cag human cd63 gfp/product/Lonza
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    93
    Lonza rfp gfp lc3 plasmid
    Autophagosome formation is inhibited to a greater extent in WT-MMCs compared to miR-192-KO MMCs following TGF-β treatment. ( A ) Representative confocal microscopy images showing <t>RFP-LC3</t> puncta in WT or miR-192-KO MMCs transfected with <t>GFP-RFP-LC3</t> plasmid (1 µg) followed by TGF-β treatment of these cells for 24 hrs. x1000 (10 × 100 oil) magnification. ( B ) Bar graph showing quantification of RFP-LC3 puncta in WT and miR-192-KO MMCs. *P
    Rfp Gfp Lc3 Plasmid, supplied by Lonza, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
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    94
    Lonza green fluorescent protein gfp expression plasmid
    Excision and integration activities of Sleeping Beauty <t>transposase</t> mutants. ( A ) Transposon excision assay. A genetically tagged SB transposon disrupts the <t>GFP</t> coding sequence maintained on a plasmid. Cells transfected with this construct do not express GFP. In the presence of SB transposase excision occurs, and in a fraction of the products the GFP coding sequence is restored, thereby leading to green fluorescence that can be quantified. ( B ) Relative excision efficiencies. Plasmids expressing transposase mutants were transiently cotransfected with a transposon-donor plasmid (pCMV(CAT)-GFP//T2Neo) into HeLa cells. The frequency of excision is indicated by GFP fluorescence intensity, determined by FACS analysis and normalized to SB100X. An inactive SB transposase (D3) was included as negative control. Data are represented as mean ± SD, n = 3 biological replicates. Differences in excision activities are significant as determined by Student's t-test for K248T P = 0.018, for all other mutants P
    Green Fluorescent Protein Gfp Expression Plasmid, supplied by Lonza, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Human CD63-GFP localization and extracellular vesicle (EV) analysis in the primary fibroblast cells obtained from the caudal vertebrae of Tg rats. ( a ) Localization of human CD63-GFP in the cultured Tg rat cells. Immunostaining indicated the co-localization of GFP with human CD63 (upper panels) and with rat CD63-positive signals (lower panels) around nuclei (blue). Scale bars = 50 μm. ( b ) Size distribution of the EVs isolated from the conditioned medium of Wt and Tg rat cells was determined using a NanoSight system. ( c , d ) The relationship between ceramide and the secretion of EVs. The intracellular rat CD63-positive signals and GFP signals were increased after treatment with 10 μM GW4869, a neutral sphingomyelinase (nSMase) inhibitor, for 24 hours ( c ). Western blotting showed a GW4869-dependent decrease of EV markers (rat CD63 and flotillin-1) and human CD63-GFP in the isolated EVs ( d ; left). However, the expression levels of rat CD63 and human CD63-GFP in the cell lysates were not changed by GW4869 ( d ; right). β-actin was used as a loading control. Both generation and protein composition of the EVs did not show an apparent change by CD63-GFP overexpression ( Supplementary Fig. 5 ).

    Journal: Scientific Reports

    Article Title: Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids

    doi: 10.1038/srep31172

    Figure Lengend Snippet: Human CD63-GFP localization and extracellular vesicle (EV) analysis in the primary fibroblast cells obtained from the caudal vertebrae of Tg rats. ( a ) Localization of human CD63-GFP in the cultured Tg rat cells. Immunostaining indicated the co-localization of GFP with human CD63 (upper panels) and with rat CD63-positive signals (lower panels) around nuclei (blue). Scale bars = 50 μm. ( b ) Size distribution of the EVs isolated from the conditioned medium of Wt and Tg rat cells was determined using a NanoSight system. ( c , d ) The relationship between ceramide and the secretion of EVs. The intracellular rat CD63-positive signals and GFP signals were increased after treatment with 10 μM GW4869, a neutral sphingomyelinase (nSMase) inhibitor, for 24 hours ( c ). Western blotting showed a GW4869-dependent decrease of EV markers (rat CD63 and flotillin-1) and human CD63-GFP in the isolated EVs ( d ; left). However, the expression levels of rat CD63 and human CD63-GFP in the cell lysates were not changed by GW4869 ( d ; right). β-actin was used as a loading control. Both generation and protein composition of the EVs did not show an apparent change by CD63-GFP overexpression ( Supplementary Fig. 5 ).

    Article Snippet: The plasmid CAG/human CD63-GFP was linearized by SalI and was then transfected into Wistar rESCs using a Mouse ES Cell Nucleofector Kit (Lonza, Basel, Swiss) and an Amaxa Nucleofector (A-013 program) as described previously . pCAG/human CD63-GFP contained a neomycin/kanamycin selection cassette.

    Techniques: Cell Culture, Immunostaining, Isolation, Western Blot, Expressing, Over Expression

    Human CD63-GFP expression analysis in Tg rats (Wister-esTgN(CAG/CD63-GFP)3NCCRI). ( a ) Pictures of main organs from Tg offspring (i–xiii: bright field, i’–xiii’: GFP, and xiii”: merged). GFP-negative (i and i’) and GFP-positive (ii and ii’) offspring were littermate. The heart, kidneys and stomach showed especially high fluorescent signals (xiii”). ( b ) Western blotting for endogenous rat CD63 and exogenous human CD63 in tissue lysates from GFP-negative (GFP−) and GFP-positive (GFP+) offspring. Ctx: cortex, cbl: cerebellum, and hip: hippocampus.

    Journal: Scientific Reports

    Article Title: Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids

    doi: 10.1038/srep31172

    Figure Lengend Snippet: Human CD63-GFP expression analysis in Tg rats (Wister-esTgN(CAG/CD63-GFP)3NCCRI). ( a ) Pictures of main organs from Tg offspring (i–xiii: bright field, i’–xiii’: GFP, and xiii”: merged). GFP-negative (i and i’) and GFP-positive (ii and ii’) offspring were littermate. The heart, kidneys and stomach showed especially high fluorescent signals (xiii”). ( b ) Western blotting for endogenous rat CD63 and exogenous human CD63 in tissue lysates from GFP-negative (GFP−) and GFP-positive (GFP+) offspring. Ctx: cortex, cbl: cerebellum, and hip: hippocampus.

    Article Snippet: The plasmid CAG/human CD63-GFP was linearized by SalI and was then transfected into Wistar rESCs using a Mouse ES Cell Nucleofector Kit (Lonza, Basel, Swiss) and an Amaxa Nucleofector (A-013 program) as described previously . pCAG/human CD63-GFP contained a neomycin/kanamycin selection cassette.

    Techniques: Expressing, Western Blot

    Analysis of the EVs labelled with human CD63-GFP in the three body fluids. ( a , b ) Western blotting analysis of the EVs isolated from serum ( a ), breast milk and AF ( b ) of Wt and Tg rats for flotillin-1, rat CD63, human CD63 and copGFP. AF samples were collected from pregnant Tg rats at E16–17 after mating with Wt males. GFP−: GFP-negative foetuses. GFP+: GFP-positive foetuses. ( c ) Immunoelectron microscopy images of serum-derived EVs from Wt and Tg rats using anti-human CD63 antibody (10 nm gold particles). Scale bars = 200 nm.

    Journal: Scientific Reports

    Article Title: Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids

    doi: 10.1038/srep31172

    Figure Lengend Snippet: Analysis of the EVs labelled with human CD63-GFP in the three body fluids. ( a , b ) Western blotting analysis of the EVs isolated from serum ( a ), breast milk and AF ( b ) of Wt and Tg rats for flotillin-1, rat CD63, human CD63 and copGFP. AF samples were collected from pregnant Tg rats at E16–17 after mating with Wt males. GFP−: GFP-negative foetuses. GFP+: GFP-positive foetuses. ( c ) Immunoelectron microscopy images of serum-derived EVs from Wt and Tg rats using anti-human CD63 antibody (10 nm gold particles). Scale bars = 200 nm.

    Article Snippet: The plasmid CAG/human CD63-GFP was linearized by SalI and was then transfected into Wistar rESCs using a Mouse ES Cell Nucleofector Kit (Lonza, Basel, Swiss) and an Amaxa Nucleofector (A-013 program) as described previously . pCAG/human CD63-GFP contained a neomycin/kanamycin selection cassette.

    Techniques: Western Blot, Isolation, Immuno-Electron Microscopy, Derivative Assay

    Generation of CAG/human CD63-GFP transgenic (Tg) rats. ( a ) Structure of the transgene construction. The transgene was constructed using human CD63-copGFP under control of the CAG promoter. ( b ) Image of rat embryonic stem cells (rESCs) transfected with the CAG/human CD63-GFP gene. The cultured rESCs expressed GFP. ( c ) Blastocysts after microinjection of the transfected rESCs. The arrow indicates rESC adherence to the inner cell mass (ICM). BF: bright field. Scale bars = 100 μm. ( d ) Adult female chimaeric rat from Wister-derived rESC (white-coated) injection into LEA blastocysts (brown-coated). White patches were present in the face (arrowhead). Two Tg offspring (white-coated) from mating a female chimaeric rat with a Wistar wild type (Wt) male (arrows). ( e ) Genotyping by PCR analysis of the extracted DNA from ear snips of the offspring. B: brown coat colour, W: white coat colour, V: CAG/human CD63-GFP vector, and M: size marker.

    Journal: Scientific Reports

    Article Title: Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids

    doi: 10.1038/srep31172

    Figure Lengend Snippet: Generation of CAG/human CD63-GFP transgenic (Tg) rats. ( a ) Structure of the transgene construction. The transgene was constructed using human CD63-copGFP under control of the CAG promoter. ( b ) Image of rat embryonic stem cells (rESCs) transfected with the CAG/human CD63-GFP gene. The cultured rESCs expressed GFP. ( c ) Blastocysts after microinjection of the transfected rESCs. The arrow indicates rESC adherence to the inner cell mass (ICM). BF: bright field. Scale bars = 100 μm. ( d ) Adult female chimaeric rat from Wister-derived rESC (white-coated) injection into LEA blastocysts (brown-coated). White patches were present in the face (arrowhead). Two Tg offspring (white-coated) from mating a female chimaeric rat with a Wistar wild type (Wt) male (arrows). ( e ) Genotyping by PCR analysis of the extracted DNA from ear snips of the offspring. B: brown coat colour, W: white coat colour, V: CAG/human CD63-GFP vector, and M: size marker.

    Article Snippet: The plasmid CAG/human CD63-GFP was linearized by SalI and was then transfected into Wistar rESCs using a Mouse ES Cell Nucleofector Kit (Lonza, Basel, Swiss) and an Amaxa Nucleofector (A-013 program) as described previously . pCAG/human CD63-GFP contained a neomycin/kanamycin selection cassette.

    Techniques: Transgenic Assay, Construct, Transfection, Cell Culture, Derivative Assay, Injection, Polymerase Chain Reaction, Plasmid Preparation, Marker

    Autophagosome formation is inhibited to a greater extent in WT-MMCs compared to miR-192-KO MMCs following TGF-β treatment. ( A ) Representative confocal microscopy images showing RFP-LC3 puncta in WT or miR-192-KO MMCs transfected with GFP-RFP-LC3 plasmid (1 µg) followed by TGF-β treatment of these cells for 24 hrs. x1000 (10 × 100 oil) magnification. ( B ) Bar graph showing quantification of RFP-LC3 puncta in WT and miR-192-KO MMCs. *P

    Journal: Scientific Reports

    Article Title: Reduced Autophagy by a microRNA-mediated Signaling Cascade in Diabetes-induced Renal Glomerular Hypertrophy

    doi: 10.1038/s41598-018-25295-x

    Figure Lengend Snippet: Autophagosome formation is inhibited to a greater extent in WT-MMCs compared to miR-192-KO MMCs following TGF-β treatment. ( A ) Representative confocal microscopy images showing RFP-LC3 puncta in WT or miR-192-KO MMCs transfected with GFP-RFP-LC3 plasmid (1 µg) followed by TGF-β treatment of these cells for 24 hrs. x1000 (10 × 100 oil) magnification. ( B ) Bar graph showing quantification of RFP-LC3 puncta in WT and miR-192-KO MMCs. *P

    Article Snippet: Determination of autophagosome formation MMCs were transfected with 1 µg RFP-GFP-LC3 plasmid using a Nucleofector and Basic Nucleofector Kit (Lonza).

    Techniques: Confocal Microscopy, Transfection, Plasmid Preparation

    Autophagosome formation is inhibited to a greater extent in WT-MMCs compared to miR-192-KO MMCs following TGF-β treatment. ( A ) Representative confocal microscopy images showing RFP-LC3 puncta in WT or miR-192-KO MMCs transfected with GFP-RFP-LC3 plasmid (1 µg) followed by TGF-β treatment of these cells for 24 hrs. x1000 (10 × 100 oil) magnification. ( B ) Bar graph showing quantification of RFP-LC3 puncta in WT and miR-192-KO MMCs. *P

    Journal: Scientific Reports

    Article Title: Reduced Autophagy by a microRNA-mediated Signaling Cascade in Diabetes-induced Renal Glomerular Hypertrophy

    doi: 10.1038/s41598-018-25295-x

    Figure Lengend Snippet: Autophagosome formation is inhibited to a greater extent in WT-MMCs compared to miR-192-KO MMCs following TGF-β treatment. ( A ) Representative confocal microscopy images showing RFP-LC3 puncta in WT or miR-192-KO MMCs transfected with GFP-RFP-LC3 plasmid (1 µg) followed by TGF-β treatment of these cells for 24 hrs. x1000 (10 × 100 oil) magnification. ( B ) Bar graph showing quantification of RFP-LC3 puncta in WT and miR-192-KO MMCs. *P

    Article Snippet: MMCs were transfected with 1 µg RFP-GFP-LC3 plasmid using a Nucleofector and Basic Nucleofector Kit (Lonza).

    Techniques: Confocal Microscopy, Transfection, Plasmid Preparation

    Excision and integration activities of Sleeping Beauty transposase mutants. ( A ) Transposon excision assay. A genetically tagged SB transposon disrupts the GFP coding sequence maintained on a plasmid. Cells transfected with this construct do not express GFP. In the presence of SB transposase excision occurs, and in a fraction of the products the GFP coding sequence is restored, thereby leading to green fluorescence that can be quantified. ( B ) Relative excision efficiencies. Plasmids expressing transposase mutants were transiently cotransfected with a transposon-donor plasmid (pCMV(CAT)-GFP//T2Neo) into HeLa cells. The frequency of excision is indicated by GFP fluorescence intensity, determined by FACS analysis and normalized to SB100X. An inactive SB transposase (D3) was included as negative control. Data are represented as mean ± SD, n = 3 biological replicates. Differences in excision activities are significant as determined by Student's t-test for K248T P = 0.018, for all other mutants P

    Journal: Nucleic Acids Research

    Article Title: A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming

    doi: 10.1093/nar/gkz1119

    Figure Lengend Snippet: Excision and integration activities of Sleeping Beauty transposase mutants. ( A ) Transposon excision assay. A genetically tagged SB transposon disrupts the GFP coding sequence maintained on a plasmid. Cells transfected with this construct do not express GFP. In the presence of SB transposase excision occurs, and in a fraction of the products the GFP coding sequence is restored, thereby leading to green fluorescence that can be quantified. ( B ) Relative excision efficiencies. Plasmids expressing transposase mutants were transiently cotransfected with a transposon-donor plasmid (pCMV(CAT)-GFP//T2Neo) into HeLa cells. The frequency of excision is indicated by GFP fluorescence intensity, determined by FACS analysis and normalized to SB100X. An inactive SB transposase (D3) was included as negative control. Data are represented as mean ± SD, n = 3 biological replicates. Differences in excision activities are significant as determined by Student's t-test for K248T P = 0.018, for all other mutants P

    Article Snippet: 50 ng of transposase plasmid [mutant SB100X transposase or wild-type SB100X (pCMV(CAT)T7-SB100X)] or green fluorescent protein (GFP) expression plasmid (pmaxGFP™; Lonza, Basel, Switzerland) were transfected.

    Techniques: Excision Assay, Sequencing, Plasmid Preparation, Transfection, Construct, Fluorescence, Expressing, FACS, Negative Control

    Reprogramming cassette removal with K248T in mouse induced pluripotent stem cells. ( A ) Transient transgenesis with an SB vector conta ining reprogramming genes and their removal with transient expression of the K248T SB transposase. Schematic representation of the SB transposon-based reprogramming vector pT2-CAG.OSKML-puΔtk ( 26 ). Individual genes in five-factor cassettes were linked by 2A self-cleaving peptide sequence and expressed from the CAG promoter. Black arrows represent SB transposon TIRs; pA, bovine growth hormone polyadenylation signal; puΔtk, PGK promoter-driven puΔtk expression cassette. The OSKML factors plus selection genes were removed from a single-copy mouse iPSC clone with the help of the SB100X transposase mutant K248T. ( B ) Linker-mediated PCR genotyping of a mouse iPSC clone before and after cassette removal. The lack of a PCR product in lane 2 of the agarose gel indicates that the reprogramming cassette was successfully removed with K248T (top gel). The quality of the genomic DNAs was verified by control PCR using GAPDH primers (bottom gel). ( C ) Phenotyping of a reprogramming factor-free mouse iPSC clone. Reprogramming factor-free mouse iPSCs express the endogenous nanog pluripotency marker (immunostaining) and GFP from the endogenous Oct4 promoter. Scale bar: 50 μm.

    Journal: Nucleic Acids Research

    Article Title: A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming

    doi: 10.1093/nar/gkz1119

    Figure Lengend Snippet: Reprogramming cassette removal with K248T in mouse induced pluripotent stem cells. ( A ) Transient transgenesis with an SB vector conta ining reprogramming genes and their removal with transient expression of the K248T SB transposase. Schematic representation of the SB transposon-based reprogramming vector pT2-CAG.OSKML-puΔtk ( 26 ). Individual genes in five-factor cassettes were linked by 2A self-cleaving peptide sequence and expressed from the CAG promoter. Black arrows represent SB transposon TIRs; pA, bovine growth hormone polyadenylation signal; puΔtk, PGK promoter-driven puΔtk expression cassette. The OSKML factors plus selection genes were removed from a single-copy mouse iPSC clone with the help of the SB100X transposase mutant K248T. ( B ) Linker-mediated PCR genotyping of a mouse iPSC clone before and after cassette removal. The lack of a PCR product in lane 2 of the agarose gel indicates that the reprogramming cassette was successfully removed with K248T (top gel). The quality of the genomic DNAs was verified by control PCR using GAPDH primers (bottom gel). ( C ) Phenotyping of a reprogramming factor-free mouse iPSC clone. Reprogramming factor-free mouse iPSCs express the endogenous nanog pluripotency marker (immunostaining) and GFP from the endogenous Oct4 promoter. Scale bar: 50 μm.

    Article Snippet: 50 ng of transposase plasmid [mutant SB100X transposase or wild-type SB100X (pCMV(CAT)T7-SB100X)] or green fluorescent protein (GFP) expression plasmid (pmaxGFP™; Lonza, Basel, Switzerland) were transfected.

    Techniques: Plasmid Preparation, Expressing, Sequencing, Selection, Mutagenesis, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Immunostaining