Structured Review

Polyplus Transfection plasmid dna
Overexpression of ribosomal protein S6 (rpS6) constitutively active quadruple phosphomimetic mutant (p-rpS6-MT) vs. wild-type rpS6 (rpS6-WT) and empty vector control (pCI-neo/Ctrl) in the testis in vivo effectively impairs spermatogenesis. A : regimen used for overexpressing rpS6-WT (i.e., the full-length rpS6 cDNA) and p-rpS6-MT. <t>Transfection</t> was performed with 15 µg plasmid <t>DNA</t> per testis using Polyplus in vivo-jetPEI transfection reagent. B : immunoblot analysis revealed that overexpression of empty vector (pCI-neo) alone had no effects on the steady-state level of proteins in Sertoli cells compared with normal adult rat testes. Results are representative findings of n = 3 rats. β-Actin served as the protein loading control. C : overexpression of target proteins, namely, rpS6, p-rpS6 S235/S236, and p-rpS6 S240/S244, in testes overexpressed with rpS6-WT and p-rpS6-MT vs. pCI-neo/Ctrl was confirmed by immunoblotting. Actin served as the protein loading control. Results were representative findings of n = 3 rats. D : hematoxylin and eosin staining of paraffin cross sections of testes. In the control group (testes transfected with pCI-neo empty vector), elongating/elongated spermatids were aligned in an organized manner; representative tubules at stages V, VIII, and IX as well as the enlarged images boxed in green and blue are shown. For instance, polarized step 19 spermatids with their heads pointed to the basement membrane aligned almost perpendicular to the basement membrane, and phagosomes (annotated by yellow arrowheads) transported to the base of the seminiferous tubule are shown in a stage VIII and stage IX tubule, respectively. Tubules from testes following overexpression with rpS6-WT had extensive defects in spermatogenesis, including 1 )], 2 ) spermatids with defects in polarity (in which the heads of these spermatids were misoriented by pointing 90–180° away from the basement membrane; red arrowheads), 3 ) elongated spermatids that were trapped deep inside the seminiferous epithelium (blue arrowhead) after spermiation in a stage IX tubule; and 4 ) phagosomes (yellow arrowhead) persistently found near the tubule lumen when they should have been transported to the base of the tubule in stage IX tubules. Following p-rpS6-MT overexpression, considerably more tubules displayed defects in spermatogenesis vs. rpS6-WT. Defects to spermatogenesis noted in p-rpS6-MT-overexpressed testes are annotated by arrowheads corresponding to those in the rpS6-WT group. Micrographs shown herein are representative findings of an experimental set, which was repeated four times with testes from n = 4 rats and yielded similar results. Scale bar in low- and high-magnification images, 180 and 60 µm, respectively (except scale bar in panel at bottom of p-rpS6-MT group, 100 µm); scale bar in inset , 30 µm; these apply to corresponding images.
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1) Product Images from "mTORC1/rpS6 regulates blood-testis barrier dynamics and spermatogenetic function in the testis in vivo"

Article Title: mTORC1/rpS6 regulates blood-testis barrier dynamics and spermatogenetic function in the testis in vivo

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00263.2017

Overexpression of ribosomal protein S6 (rpS6) constitutively active quadruple phosphomimetic mutant (p-rpS6-MT) vs. wild-type rpS6 (rpS6-WT) and empty vector control (pCI-neo/Ctrl) in the testis in vivo effectively impairs spermatogenesis. A : regimen used for overexpressing rpS6-WT (i.e., the full-length rpS6 cDNA) and p-rpS6-MT. Transfection was performed with 15 µg plasmid DNA per testis using Polyplus in vivo-jetPEI transfection reagent. B : immunoblot analysis revealed that overexpression of empty vector (pCI-neo) alone had no effects on the steady-state level of proteins in Sertoli cells compared with normal adult rat testes. Results are representative findings of n = 3 rats. β-Actin served as the protein loading control. C : overexpression of target proteins, namely, rpS6, p-rpS6 S235/S236, and p-rpS6 S240/S244, in testes overexpressed with rpS6-WT and p-rpS6-MT vs. pCI-neo/Ctrl was confirmed by immunoblotting. Actin served as the protein loading control. Results were representative findings of n = 3 rats. D : hematoxylin and eosin staining of paraffin cross sections of testes. In the control group (testes transfected with pCI-neo empty vector), elongating/elongated spermatids were aligned in an organized manner; representative tubules at stages V, VIII, and IX as well as the enlarged images boxed in green and blue are shown. For instance, polarized step 19 spermatids with their heads pointed to the basement membrane aligned almost perpendicular to the basement membrane, and phagosomes (annotated by yellow arrowheads) transported to the base of the seminiferous tubule are shown in a stage VIII and stage IX tubule, respectively. Tubules from testes following overexpression with rpS6-WT had extensive defects in spermatogenesis, including 1 )], 2 ) spermatids with defects in polarity (in which the heads of these spermatids were misoriented by pointing 90–180° away from the basement membrane; red arrowheads), 3 ) elongated spermatids that were trapped deep inside the seminiferous epithelium (blue arrowhead) after spermiation in a stage IX tubule; and 4 ) phagosomes (yellow arrowhead) persistently found near the tubule lumen when they should have been transported to the base of the tubule in stage IX tubules. Following p-rpS6-MT overexpression, considerably more tubules displayed defects in spermatogenesis vs. rpS6-WT. Defects to spermatogenesis noted in p-rpS6-MT-overexpressed testes are annotated by arrowheads corresponding to those in the rpS6-WT group. Micrographs shown herein are representative findings of an experimental set, which was repeated four times with testes from n = 4 rats and yielded similar results. Scale bar in low- and high-magnification images, 180 and 60 µm, respectively (except scale bar in panel at bottom of p-rpS6-MT group, 100 µm); scale bar in inset , 30 µm; these apply to corresponding images.
Figure Legend Snippet: Overexpression of ribosomal protein S6 (rpS6) constitutively active quadruple phosphomimetic mutant (p-rpS6-MT) vs. wild-type rpS6 (rpS6-WT) and empty vector control (pCI-neo/Ctrl) in the testis in vivo effectively impairs spermatogenesis. A : regimen used for overexpressing rpS6-WT (i.e., the full-length rpS6 cDNA) and p-rpS6-MT. Transfection was performed with 15 µg plasmid DNA per testis using Polyplus in vivo-jetPEI transfection reagent. B : immunoblot analysis revealed that overexpression of empty vector (pCI-neo) alone had no effects on the steady-state level of proteins in Sertoli cells compared with normal adult rat testes. Results are representative findings of n = 3 rats. β-Actin served as the protein loading control. C : overexpression of target proteins, namely, rpS6, p-rpS6 S235/S236, and p-rpS6 S240/S244, in testes overexpressed with rpS6-WT and p-rpS6-MT vs. pCI-neo/Ctrl was confirmed by immunoblotting. Actin served as the protein loading control. Results were representative findings of n = 3 rats. D : hematoxylin and eosin staining of paraffin cross sections of testes. In the control group (testes transfected with pCI-neo empty vector), elongating/elongated spermatids were aligned in an organized manner; representative tubules at stages V, VIII, and IX as well as the enlarged images boxed in green and blue are shown. For instance, polarized step 19 spermatids with their heads pointed to the basement membrane aligned almost perpendicular to the basement membrane, and phagosomes (annotated by yellow arrowheads) transported to the base of the seminiferous tubule are shown in a stage VIII and stage IX tubule, respectively. Tubules from testes following overexpression with rpS6-WT had extensive defects in spermatogenesis, including 1 )], 2 ) spermatids with defects in polarity (in which the heads of these spermatids were misoriented by pointing 90–180° away from the basement membrane; red arrowheads), 3 ) elongated spermatids that were trapped deep inside the seminiferous epithelium (blue arrowhead) after spermiation in a stage IX tubule; and 4 ) phagosomes (yellow arrowhead) persistently found near the tubule lumen when they should have been transported to the base of the tubule in stage IX tubules. Following p-rpS6-MT overexpression, considerably more tubules displayed defects in spermatogenesis vs. rpS6-WT. Defects to spermatogenesis noted in p-rpS6-MT-overexpressed testes are annotated by arrowheads corresponding to those in the rpS6-WT group. Micrographs shown herein are representative findings of an experimental set, which was repeated four times with testes from n = 4 rats and yielded similar results. Scale bar in low- and high-magnification images, 180 and 60 µm, respectively (except scale bar in panel at bottom of p-rpS6-MT group, 100 µm); scale bar in inset , 30 µm; these apply to corresponding images.

Techniques Used: Over Expression, Mutagenesis, Plasmid Preparation, In Vivo, Transfection, Staining

2) Product Images from "Sequestration of host metabolism by an intracellular pathogen"

Article Title: Sequestration of host metabolism by an intracellular pathogen

Journal: eLife

doi: 10.7554/eLife.12552

Flag-GlgA is imported into the inclusion lumen and enhances luminal glycogen accumulation. ( A ) HeLa cells were transfected (top right) or not (top left) with Flag-GlgA before infection, and fixed 24 hpi. PAS staining revealed an increase of intraluminal glycogen (arrowheads) in Flag-GlgA expressing cells. C. trachomatis has a 8 kb plasmid, and its loss is associated with decreased GlgA expression and impaired glycogen accumulation ( Carlson et al., 2008 ) (bottom left). Remarkably, transfection of Flag-GlgA restored luminal glycogen accumulation in cells infected with the plasmid-less strain LGV 25667R (bottom right). ( B ) Immunofluorescence on Flag-GlgA transfected cells infected with the wild-type LGV strain. DNA is stained in blue, Flag-GlgA in green and the inclusion membrane in red (anti-Cap1). Flag-GlgA is abundant in the host cytoplasm and the inclusion lumen (see also xz (top) and yz (right) projections). Scale bars: 10 µm. DOI: http://dx.doi.org/10.7554/eLife.12552.016
Figure Legend Snippet: Flag-GlgA is imported into the inclusion lumen and enhances luminal glycogen accumulation. ( A ) HeLa cells were transfected (top right) or not (top left) with Flag-GlgA before infection, and fixed 24 hpi. PAS staining revealed an increase of intraluminal glycogen (arrowheads) in Flag-GlgA expressing cells. C. trachomatis has a 8 kb plasmid, and its loss is associated with decreased GlgA expression and impaired glycogen accumulation ( Carlson et al., 2008 ) (bottom left). Remarkably, transfection of Flag-GlgA restored luminal glycogen accumulation in cells infected with the plasmid-less strain LGV 25667R (bottom right). ( B ) Immunofluorescence on Flag-GlgA transfected cells infected with the wild-type LGV strain. DNA is stained in blue, Flag-GlgA in green and the inclusion membrane in red (anti-Cap1). Flag-GlgA is abundant in the host cytoplasm and the inclusion lumen (see also xz (top) and yz (right) projections). Scale bars: 10 µm. DOI: http://dx.doi.org/10.7554/eLife.12552.016

Techniques Used: Transfection, Infection, Staining, Expressing, Plasmid Preparation, Immunofluorescence

3) Product Images from "Neuronal non-CG methylation is an essential target for MeCP2 function"

Article Title: Neuronal non-CG methylation is an essential target for MeCP2 function

Journal: bioRxiv

doi: 10.1101/2020.07.02.184614

Chimeric protein MM2 has the DNA binding properties of MBD2 in the context of MeCP2 A. Alignment of human (h) and mouse (m) MeCP2, MBD2, MBD1 and MBD4, shaded by percentage identity, with conservation below. The RTT-causing missense mutations that lie in the MBD are shown in red (above). The conserved arginine at position 111 in MeCP2 is boxed in red. The sequences from mMeCP2 and mMBD2 used in MM2 are shaded in orange. B. Schematic showing the design of the chimeric protein MM2, where the MBD of MBD2 (residues 153-217) was inserted into the MeCP2 protein sequence to replace the endogenous MBD (residues 94-164). MM2 was tagged at the C-terminus with EGFP, connected with a short peptide linker. C. EMSA analysis of the MBD of human MBD2 binding to DNA probes containing a single unmethylated or methylated CG, CAC or CAT site. MBD2[MBD] protein concentration: 2 μM. Quantification is shown below. D. EMSA analysis of the binding affinities of N-terminal fragments (residues 1-205) of WT MeCP2 and MM2 to DNA probes containing a single mCG or mCAC site. The concentrations of each protein range from 0-4.5 μM. Quantification is shown below. E. Representative images showing localization of EGFP-tagged MeCP2 (WT) and MM2 proteins to mCG-rich heterochromatic foci when they are transiently overexpressed in mouse fibroblasts (NIH-3T3). Mutation of the MBD (R111G and the equivalent mutation in MM2) abolished binding, resulting in diffuse localisation. Scale bar, 10 μm. F. FRAP analysis at heterochromatic foci of WT-EGFP and MM2-EGFP. Total numbers of cells analysed: WT-EGFP, n = 27; MM2-EGFP, n = 28; over three independent transfection experiments. Graph shows fluorescence relative to prebleach, mean ± S.E.M. Half lives: WT-EGFP = 34.66 ± 2.32 s; MM2-EGFP = 20.46 ± 2.88 s (mean ± S.E.M.). Half-lives were compared using a Mann-Whitney test: **** P
Figure Legend Snippet: Chimeric protein MM2 has the DNA binding properties of MBD2 in the context of MeCP2 A. Alignment of human (h) and mouse (m) MeCP2, MBD2, MBD1 and MBD4, shaded by percentage identity, with conservation below. The RTT-causing missense mutations that lie in the MBD are shown in red (above). The conserved arginine at position 111 in MeCP2 is boxed in red. The sequences from mMeCP2 and mMBD2 used in MM2 are shaded in orange. B. Schematic showing the design of the chimeric protein MM2, where the MBD of MBD2 (residues 153-217) was inserted into the MeCP2 protein sequence to replace the endogenous MBD (residues 94-164). MM2 was tagged at the C-terminus with EGFP, connected with a short peptide linker. C. EMSA analysis of the MBD of human MBD2 binding to DNA probes containing a single unmethylated or methylated CG, CAC or CAT site. MBD2[MBD] protein concentration: 2 μM. Quantification is shown below. D. EMSA analysis of the binding affinities of N-terminal fragments (residues 1-205) of WT MeCP2 and MM2 to DNA probes containing a single mCG or mCAC site. The concentrations of each protein range from 0-4.5 μM. Quantification is shown below. E. Representative images showing localization of EGFP-tagged MeCP2 (WT) and MM2 proteins to mCG-rich heterochromatic foci when they are transiently overexpressed in mouse fibroblasts (NIH-3T3). Mutation of the MBD (R111G and the equivalent mutation in MM2) abolished binding, resulting in diffuse localisation. Scale bar, 10 μm. F. FRAP analysis at heterochromatic foci of WT-EGFP and MM2-EGFP. Total numbers of cells analysed: WT-EGFP, n = 27; MM2-EGFP, n = 28; over three independent transfection experiments. Graph shows fluorescence relative to prebleach, mean ± S.E.M. Half lives: WT-EGFP = 34.66 ± 2.32 s; MM2-EGFP = 20.46 ± 2.88 s (mean ± S.E.M.). Half-lives were compared using a Mann-Whitney test: **** P

Techniques Used: Binding Assay, Sequencing, Methylation, Protein Concentration, Mutagenesis, Transfection, Fluorescence, MANN-WHITNEY

4) Product Images from "mTORC1/rpS6 regulates blood-testis barrier dynamics and spermatogenetic function in the testis in vivo"

Article Title: mTORC1/rpS6 regulates blood-testis barrier dynamics and spermatogenetic function in the testis in vivo

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00263.2017

Overexpression of ribosomal protein S6 (rpS6) constitutively active quadruple phosphomimetic mutant (p-rpS6-MT) vs. wild-type rpS6 (rpS6-WT) and empty vector control (pCI-neo/Ctrl) in the testis in vivo effectively impairs spermatogenesis. A : regimen used for overexpressing rpS6-WT (i.e., the full-length rpS6 cDNA) and p-rpS6-MT. Transfection was performed with 15 µg plasmid DNA per testis using Polyplus in vivo-jetPEI transfection reagent. B : immunoblot analysis revealed that overexpression of empty vector (pCI-neo) alone had no effects on the steady-state level of proteins in Sertoli cells compared with normal adult rat testes. Results are representative findings of n = 3 rats. β-Actin served as the protein loading control. C : overexpression of target proteins, namely, rpS6, p-rpS6 S235/S236, and p-rpS6 S240/S244, in testes overexpressed with rpS6-WT and p-rpS6-MT vs. pCI-neo/Ctrl was confirmed by immunoblotting. Actin served as the protein loading control. Results were representative findings of n = 3 rats. D : hematoxylin and eosin staining of paraffin cross sections of testes. In the control group (testes transfected with pCI-neo empty vector), elongating/elongated spermatids were aligned in an organized manner; representative tubules at stages V, VIII, and IX as well as the enlarged images boxed in green and blue are shown. For instance, polarized step 19 spermatids with their heads pointed to the basement membrane aligned almost perpendicular to the basement membrane, and phagosomes (annotated by yellow arrowheads) transported to the base of the seminiferous tubule are shown in a stage VIII and stage IX tubule, respectively. Tubules from testes following overexpression with rpS6-WT had extensive defects in spermatogenesis, including 1 )], 2 ) spermatids with defects in polarity (in which the heads of these spermatids were misoriented by pointing 90–180° away from the basement membrane; red arrowheads), 3 ) elongated spermatids that were trapped deep inside the seminiferous epithelium (blue arrowhead) after spermiation in a stage IX tubule; and 4 ) phagosomes (yellow arrowhead) persistently found near the tubule lumen when they should have been transported to the base of the tubule in stage IX tubules. Following p-rpS6-MT overexpression, considerably more tubules displayed defects in spermatogenesis vs. rpS6-WT. Defects to spermatogenesis noted in p-rpS6-MT-overexpressed testes are annotated by arrowheads corresponding to those in the rpS6-WT group. Micrographs shown herein are representative findings of an experimental set, which was repeated four times with testes from n = 4 rats and yielded similar results. Scale bar in low- and high-magnification images, 180 and 60 µm, respectively (except scale bar in panel at bottom of p-rpS6-MT group, 100 µm); scale bar in inset , 30 µm; these apply to corresponding images.
Figure Legend Snippet: Overexpression of ribosomal protein S6 (rpS6) constitutively active quadruple phosphomimetic mutant (p-rpS6-MT) vs. wild-type rpS6 (rpS6-WT) and empty vector control (pCI-neo/Ctrl) in the testis in vivo effectively impairs spermatogenesis. A : regimen used for overexpressing rpS6-WT (i.e., the full-length rpS6 cDNA) and p-rpS6-MT. Transfection was performed with 15 µg plasmid DNA per testis using Polyplus in vivo-jetPEI transfection reagent. B : immunoblot analysis revealed that overexpression of empty vector (pCI-neo) alone had no effects on the steady-state level of proteins in Sertoli cells compared with normal adult rat testes. Results are representative findings of n = 3 rats. β-Actin served as the protein loading control. C : overexpression of target proteins, namely, rpS6, p-rpS6 S235/S236, and p-rpS6 S240/S244, in testes overexpressed with rpS6-WT and p-rpS6-MT vs. pCI-neo/Ctrl was confirmed by immunoblotting. Actin served as the protein loading control. Results were representative findings of n = 3 rats. D : hematoxylin and eosin staining of paraffin cross sections of testes. In the control group (testes transfected with pCI-neo empty vector), elongating/elongated spermatids were aligned in an organized manner; representative tubules at stages V, VIII, and IX as well as the enlarged images boxed in green and blue are shown. For instance, polarized step 19 spermatids with their heads pointed to the basement membrane aligned almost perpendicular to the basement membrane, and phagosomes (annotated by yellow arrowheads) transported to the base of the seminiferous tubule are shown in a stage VIII and stage IX tubule, respectively. Tubules from testes following overexpression with rpS6-WT had extensive defects in spermatogenesis, including 1 )], 2 ) spermatids with defects in polarity (in which the heads of these spermatids were misoriented by pointing 90–180° away from the basement membrane; red arrowheads), 3 ) elongated spermatids that were trapped deep inside the seminiferous epithelium (blue arrowhead) after spermiation in a stage IX tubule; and 4 ) phagosomes (yellow arrowhead) persistently found near the tubule lumen when they should have been transported to the base of the tubule in stage IX tubules. Following p-rpS6-MT overexpression, considerably more tubules displayed defects in spermatogenesis vs. rpS6-WT. Defects to spermatogenesis noted in p-rpS6-MT-overexpressed testes are annotated by arrowheads corresponding to those in the rpS6-WT group. Micrographs shown herein are representative findings of an experimental set, which was repeated four times with testes from n = 4 rats and yielded similar results. Scale bar in low- and high-magnification images, 180 and 60 µm, respectively (except scale bar in panel at bottom of p-rpS6-MT group, 100 µm); scale bar in inset , 30 µm; these apply to corresponding images.

Techniques Used: Over Expression, Mutagenesis, Plasmid Preparation, In Vivo, Transfection, Staining

5) Product Images from "The Putative Type III Secreted Chlamydia abortus Virulence-Associated Protein CAB063 Targets Lamin and Induces Apoptosis"

Article Title: The Putative Type III Secreted Chlamydia abortus Virulence-Associated Protein CAB063 Targets Lamin and Induces Apoptosis

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2020.01059

CAB063 co-localizes with lamin A along the host cell nuclear membrane. Co-localization (arrows) of CAB063 and lamin A is shown in pCI-CAB063 transfected and experimentally infected HeLa cells at 48 h post-infection and post-transfection, respectively. Goat-anti mouse AlexaFluor ® 647-conjugated secondary antibodies were used to visualize mouse-anti lamin A/C antibodies (pink), goat-anti rabbit AlexaFluor ® 488-conjugated secondary antibodies were used to visualize rabbit-anti CAB063 (green), DAPI was used for DNA staining (blue). Uninfected HeLa cells served as a control. Representative photographs out of n = 3 independent experiments are provided.
Figure Legend Snippet: CAB063 co-localizes with lamin A along the host cell nuclear membrane. Co-localization (arrows) of CAB063 and lamin A is shown in pCI-CAB063 transfected and experimentally infected HeLa cells at 48 h post-infection and post-transfection, respectively. Goat-anti mouse AlexaFluor ® 647-conjugated secondary antibodies were used to visualize mouse-anti lamin A/C antibodies (pink), goat-anti rabbit AlexaFluor ® 488-conjugated secondary antibodies were used to visualize rabbit-anti CAB063 (green), DAPI was used for DNA staining (blue). Uninfected HeLa cells served as a control. Representative photographs out of n = 3 independent experiments are provided.

Techniques Used: Transfection, Infection, Staining

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Article Snippet: For siRNA-mediated knockdown in HEK293T or HeLa cells, reverse transfection using the same reagent was performed and cells were harvested after 96 h. For Large-scale transfection with plasmids for transient overexpression of proteins in HEK293T or. eGFP-Rab35endo KI HeLa cells for biochemical analysis, calcium phosphate transfection was used. .. For rescue experiments upon siRNA-mediated protein depletion, HEK293T cells were transfected with plasmid-DNA 48 h after reverse transfection with siRNA and harvested after transient overexpression for 48 h. .. Preparation of lysates from cell cultures Mammalian cell cultures were rinsed once with ice-cold PBS before cell lysis buffer (20 mM HEPES pH 7.4, 100 mM KCl, 2 mM MgCl2 , 1 % (v/v) Triton X-100) freshly supplemented with 1 mM PMSF, 0.3% protease inhibitor cocktail (Sigma) and phosphatase inhibitors (coctails 2 and 3, Sigma) was applied.

Article Title: CIP2A interacts with TopBP1 and is selectively essential for DNA damage-induced basal-like breast cancer tumorigenesis
Article Snippet: After transduction with lentiviral particles, successfully transfected (GFP positive) cells were sorted using SH800 Cell Sorter (Sony). .. Plasmid DNAs and siRNAs were transfected using Jet Prime (Polyplus Transfection) and Oligofectamine (Thermo Fisher Scientific) reagents respectively as per manufacturer’s protocols. .. DNAs were transfected for 48 hours and siRNAs were transfected for 48 to 72 hours until use for further experiments.

Article Title: Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells
Article Snippet: Briefly, the cells were seeded onto a solidified layer of Matrigel and cultured in DMEM/Ham's F-12 supplemented with 2% horse serum, 5 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin, and 2% Matrigel. .. Plasmid DNAs were transfected using jetPEI DNA transfection reagent (Polyplus transfection; Illkirch-Graffenstaden, France). siRNAs were transfected into cells using Lipofectamine RNAiMAX (Life Technologies). .. The siRNAs were used at a final concentration of 5 nM.

Article Title: Regulation of Programmed Ribosomal Frameshifting by Co-Translational Refolding RNA Hairpins
Article Snippet: One day before the transfection, 0.5–1×105 HEK-293T cells per well were placed in a 24-well culture plate with 1000 μl growth medium. .. Transfection was conducted by adding the mixture of 0.5 μg plasmid DNA and jetPEI™ transfection reagent (Polyplus) into each well, according to the manufacturer’s instructions. .. Luciferase activity measurements for transfected 293T cell lysates were performed using the Dual Luciferase™ reporter assay (Promega) according to the manufacturer’s instructions on a CHAMELEON™ multi-label plate reader (HIDEX).

Variant Assay:

Article Title: The I–II Loop Controls Plasma Membrane Expression and Gating of Cav3.2 T-Type Ca2+ Channels: A Paradigm for Childhood Absence Epilepsy Mutations
Article Snippet: HEK 293 cells (CRL-1573; American Type Culture Collection, Manassas, VA) were grown in DMEM/F-12 (Invitrogen) supplemented with 10% fetal calf serum, penicillin G (100 U/ml), and streptomycin (0.1 mg/ml). .. Cells were transiently transfected with plasmid DNAs encoding each Cav 3.2 variant using JET-PEI (Polyplus, Illkirch, France). ..

Infection:

Article Title: ERα activity depends on interaction and target site corecruitment with phosphorylated CREB1
Article Snippet: For siCREB1-mediated knockdowns, MDA-MB-134 cells were transfected with a final concentration of 20 nM siRNA, purchased as a pool of four individual siRNAs targeting CREB1 (ON-TARGETplus Human CREB1 siRNA) and a pool of four siRNAs designed as negative control (ON-TARGETplus Non-targeting Pool siRNA) from Dharmacon. .. Where indicated, siRNAs were cotransfected with plasmid DNAs; transfections were performed using Jet-Prime (PolyPlus) in medium without serum and P/S, supplemented with glutamine for 4 h. After infection and selection, or siRNA/plasmid transfection, the medium was replaced with phenol red– and serum-free medium, and the cells were incubated for 24 h before performing experiments (such as inducing cells for 24 h for luciferase or apoptosis assays). .. Cells were washed once with ice-cold PBS and lysed with a buffer containing 10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 10 mM Na-molybdate, and a protease inhibitor cocktail (Thermo Fisher Scientific).

Selection:

Article Title: ERα activity depends on interaction and target site corecruitment with phosphorylated CREB1
Article Snippet: For siCREB1-mediated knockdowns, MDA-MB-134 cells were transfected with a final concentration of 20 nM siRNA, purchased as a pool of four individual siRNAs targeting CREB1 (ON-TARGETplus Human CREB1 siRNA) and a pool of four siRNAs designed as negative control (ON-TARGETplus Non-targeting Pool siRNA) from Dharmacon. .. Where indicated, siRNAs were cotransfected with plasmid DNAs; transfections were performed using Jet-Prime (PolyPlus) in medium without serum and P/S, supplemented with glutamine for 4 h. After infection and selection, or siRNA/plasmid transfection, the medium was replaced with phenol red– and serum-free medium, and the cells were incubated for 24 h before performing experiments (such as inducing cells for 24 h for luciferase or apoptosis assays). .. Cells were washed once with ice-cold PBS and lysed with a buffer containing 10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 10 mM Na-molybdate, and a protease inhibitor cocktail (Thermo Fisher Scientific).

Incubation:

Article Title: ERα activity depends on interaction and target site corecruitment with phosphorylated CREB1
Article Snippet: For siCREB1-mediated knockdowns, MDA-MB-134 cells were transfected with a final concentration of 20 nM siRNA, purchased as a pool of four individual siRNAs targeting CREB1 (ON-TARGETplus Human CREB1 siRNA) and a pool of four siRNAs designed as negative control (ON-TARGETplus Non-targeting Pool siRNA) from Dharmacon. .. Where indicated, siRNAs were cotransfected with plasmid DNAs; transfections were performed using Jet-Prime (PolyPlus) in medium without serum and P/S, supplemented with glutamine for 4 h. After infection and selection, or siRNA/plasmid transfection, the medium was replaced with phenol red– and serum-free medium, and the cells were incubated for 24 h before performing experiments (such as inducing cells for 24 h for luciferase or apoptosis assays). .. Cells were washed once with ice-cold PBS and lysed with a buffer containing 10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 10 mM Na-molybdate, and a protease inhibitor cocktail (Thermo Fisher Scientific).

Luciferase:

Article Title: ERα activity depends on interaction and target site corecruitment with phosphorylated CREB1
Article Snippet: For siCREB1-mediated knockdowns, MDA-MB-134 cells were transfected with a final concentration of 20 nM siRNA, purchased as a pool of four individual siRNAs targeting CREB1 (ON-TARGETplus Human CREB1 siRNA) and a pool of four siRNAs designed as negative control (ON-TARGETplus Non-targeting Pool siRNA) from Dharmacon. .. Where indicated, siRNAs were cotransfected with plasmid DNAs; transfections were performed using Jet-Prime (PolyPlus) in medium without serum and P/S, supplemented with glutamine for 4 h. After infection and selection, or siRNA/plasmid transfection, the medium was replaced with phenol red– and serum-free medium, and the cells were incubated for 24 h before performing experiments (such as inducing cells for 24 h for luciferase or apoptosis assays). .. Cells were washed once with ice-cold PBS and lysed with a buffer containing 10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 10 mM Na-molybdate, and a protease inhibitor cocktail (Thermo Fisher Scientific).

Over Expression:

Article Title: Rab35-regulated lipid turnover by myotubularins represses mTORC1 activity and controls myelin growth
Article Snippet: For siRNA-mediated knockdown in HEK293T or HeLa cells, reverse transfection using the same reagent was performed and cells were harvested after 96 h. For Large-scale transfection with plasmids for transient overexpression of proteins in HEK293T or. eGFP-Rab35endo KI HeLa cells for biochemical analysis, calcium phosphate transfection was used. .. For rescue experiments upon siRNA-mediated protein depletion, HEK293T cells were transfected with plasmid-DNA 48 h after reverse transfection with siRNA and harvested after transient overexpression for 48 h. .. Preparation of lysates from cell cultures Mammalian cell cultures were rinsed once with ice-cold PBS before cell lysis buffer (20 mM HEPES pH 7.4, 100 mM KCl, 2 mM MgCl2 , 1 % (v/v) Triton X-100) freshly supplemented with 1 mM PMSF, 0.3% protease inhibitor cocktail (Sigma) and phosphatase inhibitors (coctails 2 and 3, Sigma) was applied.

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    Polyplus Transfection plasmid dna
    Dub3 inhibits RNF8 and RNF168 function and restrains RNF168 recruitment to sites of <t>DNA</t> lesions. (A) 293T cells were <t>transfected</t> with the indicated plasmids. The next day, cells were treated with HU, lysed and analysed by western blotting using the indicated
    Plasmid Dna, supplied by Polyplus Transfection, 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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    Dub3 inhibits RNF8 and RNF168 function and restrains RNF168 recruitment to sites of DNA lesions. (A) 293T cells were transfected with the indicated plasmids. The next day, cells were treated with HU, lysed and analysed by western blotting using the indicated

    Journal: Molecular Oncology

    Article Title: Dub3 controls DNA damage signalling by direct deubiquitination of H2AX), Dub3 controls DNA damage signalling by direct deubiquitination of H2AX

    doi: 10.1016/j.molonc.2014.03.003

    Figure Lengend Snippet: Dub3 inhibits RNF8 and RNF168 function and restrains RNF168 recruitment to sites of DNA lesions. (A) 293T cells were transfected with the indicated plasmids. The next day, cells were treated with HU, lysed and analysed by western blotting using the indicated

    Article Snippet: Plasmid DNA was transfected into cells using the calcium phosphate transfection method or using jetPRIME (Polyplus).

    Techniques: Transfection, Western Blot

    Overexpression of Dub3 abrogates DNA damage‐induced focus formation of 53BP1 and BRCA1, but not γH2AX and MDC1. (A) U2OS cells were transfected with GFP‐Dub3 WT or CI. Cells were left untreated or treated with IR (2Gy). 1 h

    Journal: Molecular Oncology

    Article Title: Dub3 controls DNA damage signalling by direct deubiquitination of H2AX), Dub3 controls DNA damage signalling by direct deubiquitination of H2AX

    doi: 10.1016/j.molonc.2014.03.003

    Figure Lengend Snippet: Overexpression of Dub3 abrogates DNA damage‐induced focus formation of 53BP1 and BRCA1, but not γH2AX and MDC1. (A) U2OS cells were transfected with GFP‐Dub3 WT or CI. Cells were left untreated or treated with IR (2Gy). 1 h

    Article Snippet: Plasmid DNA was transfected into cells using the calcium phosphate transfection method or using jetPRIME (Polyplus).

    Techniques: Over Expression, Transfection

    Biodistribution and expression of the gene therapy product . ( a ) CYL-02 was detected by qPCR at the time indicated in the blood of patients receiving 1,000 μg of the gene therapy product. *Indicates 6 hours postintratumoral injection of CYL-02. Data are means ± SD of four biological replicates per group with three experimental replicates and expressed as copies per ml of blood. Experimental threshold: 10 copies/ml of blood; experimental background in blood: 7.8 ± 0.2 × 10 4 copies per ml of blood. ( b ) CYL-02 DNA was detected by qPCR in the tumors of patients at 1 month following gene therapy. Data are means ± SD of four (patients receiving 250 and 1,000 μg of CYL-02) or six (patients receiving 500 μg of CYL-02) biological replicates per group with three experimental replicates and expressed as copy numbers of CYL-02 per ng of tumor DNA. For statistical comparison of two experimental groups, the bilateral Student's t -test was used (* P

    Journal: Molecular Therapy

    Article Title: First-in-man Phase 1 Clinical Trial of Gene Therapy for Advanced Pancreatic Cancer: Safety, Biodistribution, and Preliminary Clinical Findings

    doi: 10.1038/mt.2015.1

    Figure Lengend Snippet: Biodistribution and expression of the gene therapy product . ( a ) CYL-02 was detected by qPCR at the time indicated in the blood of patients receiving 1,000 μg of the gene therapy product. *Indicates 6 hours postintratumoral injection of CYL-02. Data are means ± SD of four biological replicates per group with three experimental replicates and expressed as copies per ml of blood. Experimental threshold: 10 copies/ml of blood; experimental background in blood: 7.8 ± 0.2 × 10 4 copies per ml of blood. ( b ) CYL-02 DNA was detected by qPCR in the tumors of patients at 1 month following gene therapy. Data are means ± SD of four (patients receiving 250 and 1,000 μg of CYL-02) or six (patients receiving 500 μg of CYL-02) biological replicates per group with three experimental replicates and expressed as copy numbers of CYL-02 per ng of tumor DNA. For statistical comparison of two experimental groups, the bilateral Student's t -test was used (* P

    Article Snippet: CYL-02 is a complex of plasmid DNA and linear polymers of polyethyleneimine (JetPEI 22 kDa from Polyplus Transfection, Illkirch, France), prepared in 5% w/v glucose with a PEI nitrogen to DNA phosphate (N/P) ratio of 8 to 10.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Injection

    Overexpression of ribosomal protein S6 (rpS6) constitutively active quadruple phosphomimetic mutant (p-rpS6-MT) vs. wild-type rpS6 (rpS6-WT) and empty vector control (pCI-neo/Ctrl) in the testis in vivo effectively impairs spermatogenesis. A : regimen used for overexpressing rpS6-WT (i.e., the full-length rpS6 cDNA) and p-rpS6-MT. Transfection was performed with 15 µg plasmid DNA per testis using Polyplus in vivo-jetPEI transfection reagent. B : immunoblot analysis revealed that overexpression of empty vector (pCI-neo) alone had no effects on the steady-state level of proteins in Sertoli cells compared with normal adult rat testes. Results are representative findings of n = 3 rats. β-Actin served as the protein loading control. C : overexpression of target proteins, namely, rpS6, p-rpS6 S235/S236, and p-rpS6 S240/S244, in testes overexpressed with rpS6-WT and p-rpS6-MT vs. pCI-neo/Ctrl was confirmed by immunoblotting. Actin served as the protein loading control. Results were representative findings of n = 3 rats. D : hematoxylin and eosin staining of paraffin cross sections of testes. In the control group (testes transfected with pCI-neo empty vector), elongating/elongated spermatids were aligned in an organized manner; representative tubules at stages V, VIII, and IX as well as the enlarged images boxed in green and blue are shown. For instance, polarized step 19 spermatids with their heads pointed to the basement membrane aligned almost perpendicular to the basement membrane, and phagosomes (annotated by yellow arrowheads) transported to the base of the seminiferous tubule are shown in a stage VIII and stage IX tubule, respectively. Tubules from testes following overexpression with rpS6-WT had extensive defects in spermatogenesis, including 1 )], 2 ) spermatids with defects in polarity (in which the heads of these spermatids were misoriented by pointing 90–180° away from the basement membrane; red arrowheads), 3 ) elongated spermatids that were trapped deep inside the seminiferous epithelium (blue arrowhead) after spermiation in a stage IX tubule; and 4 ) phagosomes (yellow arrowhead) persistently found near the tubule lumen when they should have been transported to the base of the tubule in stage IX tubules. Following p-rpS6-MT overexpression, considerably more tubules displayed defects in spermatogenesis vs. rpS6-WT. Defects to spermatogenesis noted in p-rpS6-MT-overexpressed testes are annotated by arrowheads corresponding to those in the rpS6-WT group. Micrographs shown herein are representative findings of an experimental set, which was repeated four times with testes from n = 4 rats and yielded similar results. Scale bar in low- and high-magnification images, 180 and 60 µm, respectively (except scale bar in panel at bottom of p-rpS6-MT group, 100 µm); scale bar in inset , 30 µm; these apply to corresponding images.

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: mTORC1/rpS6 regulates blood-testis barrier dynamics and spermatogenetic function in the testis in vivo

    doi: 10.1152/ajpendo.00263.2017

    Figure Lengend Snippet: Overexpression of ribosomal protein S6 (rpS6) constitutively active quadruple phosphomimetic mutant (p-rpS6-MT) vs. wild-type rpS6 (rpS6-WT) and empty vector control (pCI-neo/Ctrl) in the testis in vivo effectively impairs spermatogenesis. A : regimen used for overexpressing rpS6-WT (i.e., the full-length rpS6 cDNA) and p-rpS6-MT. Transfection was performed with 15 µg plasmid DNA per testis using Polyplus in vivo-jetPEI transfection reagent. B : immunoblot analysis revealed that overexpression of empty vector (pCI-neo) alone had no effects on the steady-state level of proteins in Sertoli cells compared with normal adult rat testes. Results are representative findings of n = 3 rats. β-Actin served as the protein loading control. C : overexpression of target proteins, namely, rpS6, p-rpS6 S235/S236, and p-rpS6 S240/S244, in testes overexpressed with rpS6-WT and p-rpS6-MT vs. pCI-neo/Ctrl was confirmed by immunoblotting. Actin served as the protein loading control. Results were representative findings of n = 3 rats. D : hematoxylin and eosin staining of paraffin cross sections of testes. In the control group (testes transfected with pCI-neo empty vector), elongating/elongated spermatids were aligned in an organized manner; representative tubules at stages V, VIII, and IX as well as the enlarged images boxed in green and blue are shown. For instance, polarized step 19 spermatids with their heads pointed to the basement membrane aligned almost perpendicular to the basement membrane, and phagosomes (annotated by yellow arrowheads) transported to the base of the seminiferous tubule are shown in a stage VIII and stage IX tubule, respectively. Tubules from testes following overexpression with rpS6-WT had extensive defects in spermatogenesis, including 1 )], 2 ) spermatids with defects in polarity (in which the heads of these spermatids were misoriented by pointing 90–180° away from the basement membrane; red arrowheads), 3 ) elongated spermatids that were trapped deep inside the seminiferous epithelium (blue arrowhead) after spermiation in a stage IX tubule; and 4 ) phagosomes (yellow arrowhead) persistently found near the tubule lumen when they should have been transported to the base of the tubule in stage IX tubules. Following p-rpS6-MT overexpression, considerably more tubules displayed defects in spermatogenesis vs. rpS6-WT. Defects to spermatogenesis noted in p-rpS6-MT-overexpressed testes are annotated by arrowheads corresponding to those in the rpS6-WT group. Micrographs shown herein are representative findings of an experimental set, which was repeated four times with testes from n = 4 rats and yielded similar results. Scale bar in low- and high-magnification images, 180 and 60 µm, respectively (except scale bar in panel at bottom of p-rpS6-MT group, 100 µm); scale bar in inset , 30 µm; these apply to corresponding images.

    Article Snippet: In vivo transfection was performed by intratesticular injection using 50 µl transfection solution per testis, which contained 15 µg plasmid DNA (for both treatment groups vs. control group) together with 1.8 µl Polyplus in vivo-jetPEI reagent (Illkirch, France) according to the manufacturer’s instructions with an N-to-P ratio equal to 6 essentially as earlier described ( ).

    Techniques: Over Expression, Mutagenesis, Plasmid Preparation, In Vivo, Transfection, Staining

    Design and in vitro validation of miR-SOD1, an artificial miRNA targeting human SOD1 . (a) miR-SOD1 was designed to have perfect complementary to human SOD1 , and is based on the backbone of cellular miR-155. (b) HEK293 cells were transfected with 2 μg plasmid DNA. Cells were harvested at 48 hr posttransfection and RNA was isolated. SOD1 and HPRT transcripts were quantified by RT-qPCR. Relative SOD1 expression was calculated according to the ΔΔCt method, and three biological replicates were analyzed. Data are presented as average of the replicates ± SEM. (c) HEK293 cells were transfected with 4 μg plasmid DNA and cells were harvested and lysed at 72 hr posttransfection. Equal amounts of protein were run on an SDS-PAGE and incubated with anti-SOD1 (red) and anti-beta-actin (green) antibodies. (d) Alignment showing perfect complementarity between the human (Hsa) and the marmoset (Cja) mRNA sequences at the mature miR-SOD1 site, but only partial complementarity with the endogenous mouse mRNA sequence (Mmu) in particular including two mismatches within the seed sequence at position 4 and 7. miRNA, microRNA; SOD1 , superoxide dismutase 1.

    Journal: Human Gene Therapy

    Article Title: Therapeutic rAAVrh10 Mediated SOD1 Silencing in Adult SOD1G93A Mice and Nonhuman Primates

    doi: 10.1089/hum.2015.122

    Figure Lengend Snippet: Design and in vitro validation of miR-SOD1, an artificial miRNA targeting human SOD1 . (a) miR-SOD1 was designed to have perfect complementary to human SOD1 , and is based on the backbone of cellular miR-155. (b) HEK293 cells were transfected with 2 μg plasmid DNA. Cells were harvested at 48 hr posttransfection and RNA was isolated. SOD1 and HPRT transcripts were quantified by RT-qPCR. Relative SOD1 expression was calculated according to the ΔΔCt method, and three biological replicates were analyzed. Data are presented as average of the replicates ± SEM. (c) HEK293 cells were transfected with 4 μg plasmid DNA and cells were harvested and lysed at 72 hr posttransfection. Equal amounts of protein were run on an SDS-PAGE and incubated with anti-SOD1 (red) and anti-beta-actin (green) antibodies. (d) Alignment showing perfect complementarity between the human (Hsa) and the marmoset (Cja) mRNA sequences at the mature miR-SOD1 site, but only partial complementarity with the endogenous mouse mRNA sequence (Mmu) in particular including two mismatches within the seed sequence at position 4 and 7. miRNA, microRNA; SOD1 , superoxide dismutase 1.

    Article Snippet: In vitro validation Human embryonic kidney cells (HEK293, 1E6) were transfected with 2 μg plasmid DNA (Jetprime; PolyPlus) and were harvested at 48 hr posttransfection; total RNA was isolated (Trizol; Life Technologies) and SOD1 transcripts were quantified by RT-qPCR.

    Techniques: In Vitro, Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR, Expressing, SDS Page, Incubation, Sequencing