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Polysciences inc linear pei
Effect of DNA to <t>PEI</t> ratio on transfection efficiency. 293E cells were transfected with linear ( A ) or branched ( B ) 25 <t>kDa</t> PEI at various DNA (pEGFP plasmid) concentrations as described in Materials and Methods. DNA concentrations (µg ml –1 ) used were: 0.25 (circles), 0.50 (squares), 1.0 (closed diamonds), 1.5 (triangles) and 2.0 (open diamonds). Transfection efficiencies were determined by flow cytometry analysis 72 hpt.
Linear Pei, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells"

Article Title: High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells

Journal: Nucleic Acids Research

doi:

Effect of DNA to PEI ratio on transfection efficiency. 293E cells were transfected with linear ( A ) or branched ( B ) 25 kDa PEI at various DNA (pEGFP plasmid) concentrations as described in Materials and Methods. DNA concentrations (µg ml –1 ) used were: 0.25 (circles), 0.50 (squares), 1.0 (closed diamonds), 1.5 (triangles) and 2.0 (open diamonds). Transfection efficiencies were determined by flow cytometry analysis 72 hpt.
Figure Legend Snippet: Effect of DNA to PEI ratio on transfection efficiency. 293E cells were transfected with linear ( A ) or branched ( B ) 25 kDa PEI at various DNA (pEGFP plasmid) concentrations as described in Materials and Methods. DNA concentrations (µg ml –1 ) used were: 0.25 (circles), 0.50 (squares), 1.0 (closed diamonds), 1.5 (triangles) and 2.0 (open diamonds). Transfection efficiencies were determined by flow cytometry analysis 72 hpt.

Techniques Used: Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

2) Product Images from "Development of a Single Vector System that Enhances Trans-Splicing of SMN2 Transcripts"

Article Title: Development of a Single Vector System that Enhances Trans-Splicing of SMN2 Transcripts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0003468

Intracerebroventricular 25 kDa PEI pMU3 transfections in SMA model mice increases SMN protein in the spinal cord. (A) Heterozygous (m SMN +/+, h SMN +/+, h Δ7 SMN cDNA +/+) (lane a) or homozygous (lanes b–g) (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) neonatal mice (PND 0-1) were injected with pMU3 plasmid at 10 µg (1.14×10 12 plasmid copies) with 25 kDa PEI over two ventricles (lanes c–g). Mock transfection mice are shown in lane a and b. Whole spinal cord homogenates were prepared for protein or RNA extraction 24 hours post injection. In vivo RT-PCR results for trans -spliced SMN are shown in the upper panel. Lower panel depicts controls for vector expression via northern blot of mock or treatment group spinal cord RNA loaded on nitrocellouse and developed with a radio-labeled GFP probe. (B) SMA mouse spinal cord protein was resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. (C) Intra-ventricular transfections of pM13 in SMA model mice do not alter SMN levels in the spinal cord. Homozygous (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) (lanes b,c) neonatal mice (PND 0-1) were injected with 10 µg of plasmid with 25 kDa PEI over both ventricles. Whole spinal cord homogenates were prepared for protein and resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. Mock treatment is depicted in lane a. (D) Graphical summary of injection outcomes relative to SMN protein induction western blot in mice. Error bars represent ±s.d. HET, KO, pM13 (n = 4). pMU3 (n = 5).
Figure Legend Snippet: Intracerebroventricular 25 kDa PEI pMU3 transfections in SMA model mice increases SMN protein in the spinal cord. (A) Heterozygous (m SMN +/+, h SMN +/+, h Δ7 SMN cDNA +/+) (lane a) or homozygous (lanes b–g) (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) neonatal mice (PND 0-1) were injected with pMU3 plasmid at 10 µg (1.14×10 12 plasmid copies) with 25 kDa PEI over two ventricles (lanes c–g). Mock transfection mice are shown in lane a and b. Whole spinal cord homogenates were prepared for protein or RNA extraction 24 hours post injection. In vivo RT-PCR results for trans -spliced SMN are shown in the upper panel. Lower panel depicts controls for vector expression via northern blot of mock or treatment group spinal cord RNA loaded on nitrocellouse and developed with a radio-labeled GFP probe. (B) SMA mouse spinal cord protein was resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. (C) Intra-ventricular transfections of pM13 in SMA model mice do not alter SMN levels in the spinal cord. Homozygous (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) (lanes b,c) neonatal mice (PND 0-1) were injected with 10 µg of plasmid with 25 kDa PEI over both ventricles. Whole spinal cord homogenates were prepared for protein and resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. Mock treatment is depicted in lane a. (D) Graphical summary of injection outcomes relative to SMN protein induction western blot in mice. Error bars represent ±s.d. HET, KO, pM13 (n = 4). pMU3 (n = 5).

Techniques Used: Transfection, Mouse Assay, Injection, Plasmid Preparation, RNA Extraction, In Vivo, Reverse Transcription Polymerase Chain Reaction, Expressing, Northern Blot, Labeling, SDS Page, Western Blot

3) Product Images from "High cell density transient transfection of CHO cells for TGF‐β1 expression. High cell density transient transfection of CHO cells for TGF‐β1 expression"

Article Title: High cell density transient transfection of CHO cells for TGF‐β1 expression. High cell density transient transfection of CHO cells for TGF‐β1 expression

Journal: Engineering in Life Sciences

doi: 10.1002/elsc.201800174

Transfection procedure for TGF‐β1 production. Table 2 represents the transfection process, which was started by preparing precultured cells with a low cell density inoculum (1.3 × 10 6 cells/mL). After 24 hours, cells at a high density were collected, resuspended in fresh medium and transfected by the appropriate amounts of TGF‐β1‐pDNA and PEI. The transfected culture was left for 72 hpt before harvesting. Table 3 represents the calculation of total purified protein followed by the detection of TGF‐β1 with western blot analysis; Lane 1, a protein size marker is shown. Lane 2, commercial TGF‐β1 (CHO derived) and Lane 3, the purified TGF‐β1 from transfected culture supernatant. Tag that used here are Twin‐Strep‐tag 30 aa. (3 kDa) for protein purification and tryptophan tag 30 aa.(4.4 kDa) for online protein expression monitoring
Figure Legend Snippet: Transfection procedure for TGF‐β1 production. Table 2 represents the transfection process, which was started by preparing precultured cells with a low cell density inoculum (1.3 × 10 6 cells/mL). After 24 hours, cells at a high density were collected, resuspended in fresh medium and transfected by the appropriate amounts of TGF‐β1‐pDNA and PEI. The transfected culture was left for 72 hpt before harvesting. Table 3 represents the calculation of total purified protein followed by the detection of TGF‐β1 with western blot analysis; Lane 1, a protein size marker is shown. Lane 2, commercial TGF‐β1 (CHO derived) and Lane 3, the purified TGF‐β1 from transfected culture supernatant. Tag that used here are Twin‐Strep‐tag 30 aa. (3 kDa) for protein purification and tryptophan tag 30 aa.(4.4 kDa) for online protein expression monitoring

Techniques Used: Transfection, Purification, Western Blot, Marker, Derivative Assay, Strep-tag, Protein Purification, Expressing

Related Articles

Transfection:

Article Title: High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals
Article Snippet: .. HEK 293 cells (Thermo Fisher Scientific, Waltham, MA) were transfected using linear PEI (Polysciences, Warrington, PA) and purification from the supernatant was performed using His60 beads (Clontech Laboratories, Mountain View, CA) according to the manufacturer recommendations. ..

Article Title: Self-antigen driven affinity maturation is required for pathogenic monovalent IgG4 autoantibody development
Article Snippet: .. Briefly, HEK293A were transfected with equal amounts of the heavy and the corresponding light chain plasmid using linear PEI (Polysciences Cat# 23966). ..

Article Title: High cell density transient transfection of CHO cells for TGF‐β1 expression. High cell density transient transfection of CHO cells for TGF‐β1 expression
Article Snippet: .. Linear PEI (MW 25 kDa; Polysciences GmbH, USA) in a stock solution of 1 mg/mL was used for transfection. ..

Article Title: Inhibition by Chondroitin Sulfate E Can Specify Functional Wnt/?-Catenin Signaling Thresholds in NIH3T3 Fibroblasts *
Article Snippet: .. NIH3T3 cells were transiently transfected with firefly TOPFLASH ( ) and Renilla luciferase transfection control reporter constructs using linear PEI ( M r 25,000, Polysciences, Inc.; PEI/DNA ratio of 5:1) as the transfection reagent. ..

Article Title: Development of a Single Vector System that Enhances Trans-Splicing of SMN2 Transcripts
Article Snippet: .. In vitro Transfections The transfections were performed using linear PEI (Polysciences, Inc.) to pH 8.3 with 20 µL adjustments of HCl or NaOH. ..

Article Title: Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma
Article Snippet: .. Competitive TSLP-SEAP cell binding assay HEK293T cells were transfected with pMET7-TSLPR127A/R130S -SEAP using linear PEI (Polysciences). ..

Article Title: Biphasic Role of Chondroitin Sulfate in Cardiac Differentiation of Embryonic Stem Cells through Inhibition of Wnt/?-Catenin Signaling
Article Snippet: .. TOPFLASH Reporter Assays ES cells or EBs were transiently transfected with firefly TOPFLASH and Renilla luciferase transfection control reporter constructs, using linear PEI (MW: 25,000; Polysciences, USA; PEI/DNA ratio of 8∶1) as a transfection reagent as previously described . ..

Purification:

Article Title: High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals
Article Snippet: .. HEK 293 cells (Thermo Fisher Scientific, Waltham, MA) were transfected using linear PEI (Polysciences, Warrington, PA) and purification from the supernatant was performed using His60 beads (Clontech Laboratories, Mountain View, CA) according to the manufacturer recommendations. ..

Plasmid Preparation:

Article Title: Self-antigen driven affinity maturation is required for pathogenic monovalent IgG4 autoantibody development
Article Snippet: .. Briefly, HEK293A were transfected with equal amounts of the heavy and the corresponding light chain plasmid using linear PEI (Polysciences Cat# 23966). ..

Luciferase:

Article Title: Inhibition by Chondroitin Sulfate E Can Specify Functional Wnt/?-Catenin Signaling Thresholds in NIH3T3 Fibroblasts *
Article Snippet: .. NIH3T3 cells were transiently transfected with firefly TOPFLASH ( ) and Renilla luciferase transfection control reporter constructs using linear PEI ( M r 25,000, Polysciences, Inc.; PEI/DNA ratio of 5:1) as the transfection reagent. ..

Article Title: Biphasic Role of Chondroitin Sulfate in Cardiac Differentiation of Embryonic Stem Cells through Inhibition of Wnt/?-Catenin Signaling
Article Snippet: .. TOPFLASH Reporter Assays ES cells or EBs were transiently transfected with firefly TOPFLASH and Renilla luciferase transfection control reporter constructs, using linear PEI (MW: 25,000; Polysciences, USA; PEI/DNA ratio of 8∶1) as a transfection reagent as previously described . ..

Construct:

Article Title: Inhibition by Chondroitin Sulfate E Can Specify Functional Wnt/?-Catenin Signaling Thresholds in NIH3T3 Fibroblasts *
Article Snippet: .. NIH3T3 cells were transiently transfected with firefly TOPFLASH ( ) and Renilla luciferase transfection control reporter constructs using linear PEI ( M r 25,000, Polysciences, Inc.; PEI/DNA ratio of 5:1) as the transfection reagent. ..

Article Title: Biphasic Role of Chondroitin Sulfate in Cardiac Differentiation of Embryonic Stem Cells through Inhibition of Wnt/?-Catenin Signaling
Article Snippet: .. TOPFLASH Reporter Assays ES cells or EBs were transiently transfected with firefly TOPFLASH and Renilla luciferase transfection control reporter constructs, using linear PEI (MW: 25,000; Polysciences, USA; PEI/DNA ratio of 8∶1) as a transfection reagent as previously described . ..

In Vitro:

Article Title: Development of a Single Vector System that Enhances Trans-Splicing of SMN2 Transcripts
Article Snippet: .. In vitro Transfections The transfections were performed using linear PEI (Polysciences, Inc.) to pH 8.3 with 20 µL adjustments of HCl or NaOH. ..

Cell Binding Assay:

Article Title: Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma
Article Snippet: .. Competitive TSLP-SEAP cell binding assay HEK293T cells were transfected with pMET7-TSLPR127A/R130S -SEAP using linear PEI (Polysciences). ..

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    Polysciences inc linear pei
    Effect of DNA to <t>PEI</t> ratio on transfection efficiency. 293E cells were transfected with linear ( A ) or branched ( B ) 25 <t>kDa</t> PEI at various DNA (pEGFP plasmid) concentrations as described in Materials and Methods. DNA concentrations (µg ml –1 ) used were: 0.25 (circles), 0.50 (squares), 1.0 (closed diamonds), 1.5 (triangles) and 2.0 (open diamonds). Transfection efficiencies were determined by flow cytometry analysis 72 hpt.
    Linear Pei, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/linear pei/product/Polysciences inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    linear pei - by Bioz Stars, 2021-09
    86/100 stars
      Buy from Supplier

    86
    Polysciences inc linear pei homopolymer
    Intracerebroventricular 25 <t>kDa</t> <t>PEI</t> pMU3 transfections in SMA model mice increases SMN protein in the spinal cord. (A) Heterozygous (m SMN +/+, h SMN +/+, h Δ7 SMN cDNA +/+) (lane a) or homozygous (lanes b–g) (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) neonatal mice (PND 0-1) were injected with pMU3 plasmid at 10 µg (1.14×10 12 plasmid copies) with 25 kDa PEI over two ventricles (lanes c–g). Mock transfection mice are shown in lane a and b. Whole spinal cord homogenates were prepared for protein or RNA extraction 24 hours post injection. In vivo RT-PCR results for trans -spliced SMN are shown in the upper panel. Lower panel depicts controls for vector expression via northern blot of mock or treatment group spinal cord RNA loaded on nitrocellouse and developed with a radio-labeled GFP probe. (B) SMA mouse spinal cord protein was resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. (C) Intra-ventricular transfections of pM13 in SMA model mice do not alter SMN levels in the spinal cord. Homozygous (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) (lanes b,c) neonatal mice (PND 0-1) were injected with 10 µg of plasmid with 25 kDa PEI over both ventricles. Whole spinal cord homogenates were prepared for protein and resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. Mock treatment is depicted in lane a. (D) Graphical summary of injection outcomes relative to SMN protein induction western blot in mice. Error bars represent ±s.d. HET, KO, pM13 (n = 4). pMU3 (n = 5).
    Linear Pei Homopolymer, supplied by Polysciences inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/linear pei homopolymer/product/Polysciences inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    linear pei homopolymer - by Bioz Stars, 2021-09
    86/100 stars
      Buy from Supplier

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    Effect of DNA to PEI ratio on transfection efficiency. 293E cells were transfected with linear ( A ) or branched ( B ) 25 kDa PEI at various DNA (pEGFP plasmid) concentrations as described in Materials and Methods. DNA concentrations (µg ml –1 ) used were: 0.25 (circles), 0.50 (squares), 1.0 (closed diamonds), 1.5 (triangles) and 2.0 (open diamonds). Transfection efficiencies were determined by flow cytometry analysis 72 hpt.

    Journal: Nucleic Acids Research

    Article Title: High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells

    doi:

    Figure Lengend Snippet: Effect of DNA to PEI ratio on transfection efficiency. 293E cells were transfected with linear ( A ) or branched ( B ) 25 kDa PEI at various DNA (pEGFP plasmid) concentrations as described in Materials and Methods. DNA concentrations (µg ml –1 ) used were: 0.25 (circles), 0.50 (squares), 1.0 (closed diamonds), 1.5 (triangles) and 2.0 (open diamonds). Transfection efficiencies were determined by flow cytometry analysis 72 hpt.

    Article Snippet: A 25 kDa branched PEI was obtained from Aldrich (Milwaukee, WI) and 25 kDa linear PEI from Polysciences (Warrington, PA).

    Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Cytometry

    Intracerebroventricular 25 kDa PEI pMU3 transfections in SMA model mice increases SMN protein in the spinal cord. (A) Heterozygous (m SMN +/+, h SMN +/+, h Δ7 SMN cDNA +/+) (lane a) or homozygous (lanes b–g) (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) neonatal mice (PND 0-1) were injected with pMU3 plasmid at 10 µg (1.14×10 12 plasmid copies) with 25 kDa PEI over two ventricles (lanes c–g). Mock transfection mice are shown in lane a and b. Whole spinal cord homogenates were prepared for protein or RNA extraction 24 hours post injection. In vivo RT-PCR results for trans -spliced SMN are shown in the upper panel. Lower panel depicts controls for vector expression via northern blot of mock or treatment group spinal cord RNA loaded on nitrocellouse and developed with a radio-labeled GFP probe. (B) SMA mouse spinal cord protein was resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. (C) Intra-ventricular transfections of pM13 in SMA model mice do not alter SMN levels in the spinal cord. Homozygous (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) (lanes b,c) neonatal mice (PND 0-1) were injected with 10 µg of plasmid with 25 kDa PEI over both ventricles. Whole spinal cord homogenates were prepared for protein and resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. Mock treatment is depicted in lane a. (D) Graphical summary of injection outcomes relative to SMN protein induction western blot in mice. Error bars represent ±s.d. HET, KO, pM13 (n = 4). pMU3 (n = 5).

    Journal: PLoS ONE

    Article Title: Development of a Single Vector System that Enhances Trans-Splicing of SMN2 Transcripts

    doi: 10.1371/journal.pone.0003468

    Figure Lengend Snippet: Intracerebroventricular 25 kDa PEI pMU3 transfections in SMA model mice increases SMN protein in the spinal cord. (A) Heterozygous (m SMN +/+, h SMN +/+, h Δ7 SMN cDNA +/+) (lane a) or homozygous (lanes b–g) (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) neonatal mice (PND 0-1) were injected with pMU3 plasmid at 10 µg (1.14×10 12 plasmid copies) with 25 kDa PEI over two ventricles (lanes c–g). Mock transfection mice are shown in lane a and b. Whole spinal cord homogenates were prepared for protein or RNA extraction 24 hours post injection. In vivo RT-PCR results for trans -spliced SMN are shown in the upper panel. Lower panel depicts controls for vector expression via northern blot of mock or treatment group spinal cord RNA loaded on nitrocellouse and developed with a radio-labeled GFP probe. (B) SMA mouse spinal cord protein was resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. (C) Intra-ventricular transfections of pM13 in SMA model mice do not alter SMN levels in the spinal cord. Homozygous (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) (lanes b,c) neonatal mice (PND 0-1) were injected with 10 µg of plasmid with 25 kDa PEI over both ventricles. Whole spinal cord homogenates were prepared for protein and resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. Mock treatment is depicted in lane a. (D) Graphical summary of injection outcomes relative to SMN protein induction western blot in mice. Error bars represent ±s.d. HET, KO, pM13 (n = 4). pMU3 (n = 5).

    Article Snippet: In vitro Transfections The transfections were performed using linear PEI (Polysciences, Inc.) to pH 8.3 with 20 µL adjustments of HCl or NaOH.

    Techniques: Transfection, Mouse Assay, Injection, Plasmid Preparation, RNA Extraction, In Vivo, Reverse Transcription Polymerase Chain Reaction, Expressing, Northern Blot, Labeling, SDS Page, Western Blot

    Transfection procedure for TGF‐β1 production. Table 2 represents the transfection process, which was started by preparing precultured cells with a low cell density inoculum (1.3 × 10 6 cells/mL). After 24 hours, cells at a high density were collected, resuspended in fresh medium and transfected by the appropriate amounts of TGF‐β1‐pDNA and PEI. The transfected culture was left for 72 hpt before harvesting. Table 3 represents the calculation of total purified protein followed by the detection of TGF‐β1 with western blot analysis; Lane 1, a protein size marker is shown. Lane 2, commercial TGF‐β1 (CHO derived) and Lane 3, the purified TGF‐β1 from transfected culture supernatant. Tag that used here are Twin‐Strep‐tag 30 aa. (3 kDa) for protein purification and tryptophan tag 30 aa.(4.4 kDa) for online protein expression monitoring

    Journal: Engineering in Life Sciences

    Article Title: High cell density transient transfection of CHO cells for TGF‐β1 expression. High cell density transient transfection of CHO cells for TGF‐β1 expression

    doi: 10.1002/elsc.201800174

    Figure Lengend Snippet: Transfection procedure for TGF‐β1 production. Table 2 represents the transfection process, which was started by preparing precultured cells with a low cell density inoculum (1.3 × 10 6 cells/mL). After 24 hours, cells at a high density were collected, resuspended in fresh medium and transfected by the appropriate amounts of TGF‐β1‐pDNA and PEI. The transfected culture was left for 72 hpt before harvesting. Table 3 represents the calculation of total purified protein followed by the detection of TGF‐β1 with western blot analysis; Lane 1, a protein size marker is shown. Lane 2, commercial TGF‐β1 (CHO derived) and Lane 3, the purified TGF‐β1 from transfected culture supernatant. Tag that used here are Twin‐Strep‐tag 30 aa. (3 kDa) for protein purification and tryptophan tag 30 aa.(4.4 kDa) for online protein expression monitoring

    Article Snippet: Linear PEI (MW 25 kDa; Polysciences GmbH, USA) in a stock solution of 1 mg/mL was used for transfection.

    Techniques: Transfection, Purification, Western Blot, Marker, Derivative Assay, Strep-tag, Protein Purification, Expressing

    Intracerebroventricular 25 kDa PEI pMU3 transfections in SMA model mice increases SMN protein in the spinal cord. (A) Heterozygous (m SMN +/+, h SMN +/+, h Δ7 SMN cDNA +/+) (lane a) or homozygous (lanes b–g) (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) neonatal mice (PND 0-1) were injected with pMU3 plasmid at 10 µg (1.14×10 12 plasmid copies) with 25 kDa PEI over two ventricles (lanes c–g). Mock transfection mice are shown in lane a and b. Whole spinal cord homogenates were prepared for protein or RNA extraction 24 hours post injection. In vivo RT-PCR results for trans -spliced SMN are shown in the upper panel. Lower panel depicts controls for vector expression via northern blot of mock or treatment group spinal cord RNA loaded on nitrocellouse and developed with a radio-labeled GFP probe. (B) SMA mouse spinal cord protein was resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. (C) Intra-ventricular transfections of pM13 in SMA model mice do not alter SMN levels in the spinal cord. Homozygous (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) (lanes b,c) neonatal mice (PND 0-1) were injected with 10 µg of plasmid with 25 kDa PEI over both ventricles. Whole spinal cord homogenates were prepared for protein and resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. Mock treatment is depicted in lane a. (D) Graphical summary of injection outcomes relative to SMN protein induction western blot in mice. Error bars represent ±s.d. HET, KO, pM13 (n = 4). pMU3 (n = 5).

    Journal: PLoS ONE

    Article Title: Development of a Single Vector System that Enhances Trans-Splicing of SMN2 Transcripts

    doi: 10.1371/journal.pone.0003468

    Figure Lengend Snippet: Intracerebroventricular 25 kDa PEI pMU3 transfections in SMA model mice increases SMN protein in the spinal cord. (A) Heterozygous (m SMN +/+, h SMN +/+, h Δ7 SMN cDNA +/+) (lane a) or homozygous (lanes b–g) (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) neonatal mice (PND 0-1) were injected with pMU3 plasmid at 10 µg (1.14×10 12 plasmid copies) with 25 kDa PEI over two ventricles (lanes c–g). Mock transfection mice are shown in lane a and b. Whole spinal cord homogenates were prepared for protein or RNA extraction 24 hours post injection. In vivo RT-PCR results for trans -spliced SMN are shown in the upper panel. Lower panel depicts controls for vector expression via northern blot of mock or treatment group spinal cord RNA loaded on nitrocellouse and developed with a radio-labeled GFP probe. (B) SMA mouse spinal cord protein was resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. (C) Intra-ventricular transfections of pM13 in SMA model mice do not alter SMN levels in the spinal cord. Homozygous (m SMN −/−, h SMN +/+, h Δ7 SMN cDNA +/+) (lanes b,c) neonatal mice (PND 0-1) were injected with 10 µg of plasmid with 25 kDa PEI over both ventricles. Whole spinal cord homogenates were prepared for protein and resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. Mock treatment is depicted in lane a. (D) Graphical summary of injection outcomes relative to SMN protein induction western blot in mice. Error bars represent ±s.d. HET, KO, pM13 (n = 4). pMU3 (n = 5).

    Article Snippet: Injection stock solutions contained and final volumes include: D-(+)-glucose 20% (w/v) (1 µL) (Sigma), trypan blue (0.4%) saline (1 µL) (Sigma), plasmid (≈5 µg/µL) (2 µL), 25 kDa linear PEI homopolymer (150 mM) (1 µL) (Polysciences, Inc.).

    Techniques: Transfection, Mouse Assay, Injection, Plasmid Preparation, RNA Extraction, In Vivo, Reverse Transcription Polymerase Chain Reaction, Expressing, Northern Blot, Labeling, SDS Page, Western Blot