eukaryotic expression vector pcdna3 1  (Thermo Fisher)


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    Name:
    pcDNA 3 1 Mammalian Expression Vector
    Description:
    This pcDNA 3 1 vector is designed for high level constitutive expression in a variety of mammalian cell lines It contains a Geneticin selectable marker and a reverse orientation multiple cloning site The pcDNA 3 1 Expression Vector Family Three untagged versions of pcDNA 3 1 available separately each with a different selectable marker Geneticin Zeocin or Hygromycin are for use alone or in co transfections All three vectors offer the following features • Cytomegalovirus CMV enhancer promoter for high level expression• Large multiple cloning site in either forward or reverse orientations• Bovine Growth Hormone BGH polyadenylation signal and transcription termination sequence for enhanced mRNA stability• SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen i e COS 1 and COS 7 • Ampicillin resistance gene and pUC origin for selection and maintenance in E coli
    Catalog Number:
    v79520
    Price:
    None
    Applications:
    Constitutive Expression|Mammalian Expression|Protein Biology|Protein Expression
    Category:
    DNA Vectors Clones Purified Nucleic Acids Libraries
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    Structured Review

    Thermo Fisher eukaryotic expression vector pcdna3 1
    CAPA-1 NMU-like neuropeptides signal from ASG chemosensory neurons to promote gustatory learning through NMUR-1 (a) Luminescence-based calcium mobilization assay for measuring GPCR activation. NMUR-1 is expressed in CHO cells that stably co-express the promiscuous human Gα 16 subunit, which couples receptor activation to calcium release from intracellular storage sites. Intracellular calcium levels are monitored using the calcium indicator aequorin. (b) Calcium responses of CHO cells expressing NMUR-1 are shown relative (%) to the highest value (100% activation) after normalization to the total calcium response. Cells transfected with an empty <t>pcDNA3.1</t> vector are used as a control. Error bars show S.E.M (n ≥ 6). (c) The capa-1/nlp-44 gene encodes a neuropeptide precursor that harbors three predicted peptide sequences (CAPA-1-1, -2, and -3), which are flanked by mono- and dibasic cleaving sites. Black bars indicate the positions of deletions in capa-1/nlp-44 mutant alleles used in this study. (d) Sequence comparison of C. elegans CAPA-1 neuropeptides and NmU neuropeptides from H. sapiens , D. rerio , and D. melanogaster . Conserved C-terminal features are highlighted in black. Residues with similar physicochemical properties are colored gray. (e) Mean position on the gradient through time and (g) chemotactic indices of two independent deletion mutants of the capa-1/nlp-44 neuropeptide precursor gene after mock- and NaCl-conditioning. The corresponding biased random walk and klinotaxis indices are shown on Supplementary Fig. 5a . n ≥ 25 animals per genotype. (f) Co-localization of a bicistronic GFP construct harboring the promoter, genomic DNA and 3’UTR of the capa-1 gene (capa-1p::capa-1::SL2::gfp ) with mCherry expression from the ASG-specific gcy-15 promotor 55 . The asterisk marks fluorescence in the intestine from the elt-2p::gfp co-injection marker. Scale bar, 35 µm. (h) Chemotactic behavior of NaCl-conditioned capa-1 (ok3065) animals in which wild type copies of the capa-1 gene are reintroduced under the control of its promoter sequence ( capa-1p::capa-1::SL2::gfp ). The corresponding mean population position on the gradient, biased random walk and klinotaxis indices are shown on Supplementary Fig. 5b . n ≥ 37 animals per genotype. (i) Chemotactic behavior of NaCl-conditioned animals after ASG-specific RNAi-mediated knockdown of capa-1 , achieved by expressing sense and anti-sense capa-1 sequences under the control of the ASG-specific ops-1 promoter 60 . A strain carrying only the elt-2p ::GFP co-injection marker is used as a control to exclude potential effects of the transgene selection marker. The corresponding mean population position on the gradient, biased random walk and klinotaxis indices are shown on Supplementary Fig. 5c . n ≥ 27 animals per genotype.
    This pcDNA 3 1 vector is designed for high level constitutive expression in a variety of mammalian cell lines It contains a Geneticin selectable marker and a reverse orientation multiple cloning site The pcDNA 3 1 Expression Vector Family Three untagged versions of pcDNA 3 1 available separately each with a different selectable marker Geneticin Zeocin or Hygromycin are for use alone or in co transfections All three vectors offer the following features • Cytomegalovirus CMV enhancer promoter for high level expression• Large multiple cloning site in either forward or reverse orientations• Bovine Growth Hormone BGH polyadenylation signal and transcription termination sequence for enhanced mRNA stability• SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen i e COS 1 and COS 7 • Ampicillin resistance gene and pUC origin for selection and maintenance in E coli
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    Average 99 stars, based on 152 article reviews
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    Images

    1) Product Images from "Neuromedin U signaling regulates memory retrieval of learned salt avoidance in a C. elegans gustatory circuit"

    Article Title: Neuromedin U signaling regulates memory retrieval of learned salt avoidance in a C. elegans gustatory circuit

    Journal: bioRxiv

    doi: 10.1101/683888

    CAPA-1 NMU-like neuropeptides signal from ASG chemosensory neurons to promote gustatory learning through NMUR-1 (a) Luminescence-based calcium mobilization assay for measuring GPCR activation. NMUR-1 is expressed in CHO cells that stably co-express the promiscuous human Gα 16 subunit, which couples receptor activation to calcium release from intracellular storage sites. Intracellular calcium levels are monitored using the calcium indicator aequorin. (b) Calcium responses of CHO cells expressing NMUR-1 are shown relative (%) to the highest value (100% activation) after normalization to the total calcium response. Cells transfected with an empty pcDNA3.1 vector are used as a control. Error bars show S.E.M (n ≥ 6). (c) The capa-1/nlp-44 gene encodes a neuropeptide precursor that harbors three predicted peptide sequences (CAPA-1-1, -2, and -3), which are flanked by mono- and dibasic cleaving sites. Black bars indicate the positions of deletions in capa-1/nlp-44 mutant alleles used in this study. (d) Sequence comparison of C. elegans CAPA-1 neuropeptides and NmU neuropeptides from H. sapiens , D. rerio , and D. melanogaster . Conserved C-terminal features are highlighted in black. Residues with similar physicochemical properties are colored gray. (e) Mean position on the gradient through time and (g) chemotactic indices of two independent deletion mutants of the capa-1/nlp-44 neuropeptide precursor gene after mock- and NaCl-conditioning. The corresponding biased random walk and klinotaxis indices are shown on Supplementary Fig. 5a . n ≥ 25 animals per genotype. (f) Co-localization of a bicistronic GFP construct harboring the promoter, genomic DNA and 3’UTR of the capa-1 gene (capa-1p::capa-1::SL2::gfp ) with mCherry expression from the ASG-specific gcy-15 promotor 55 . The asterisk marks fluorescence in the intestine from the elt-2p::gfp co-injection marker. Scale bar, 35 µm. (h) Chemotactic behavior of NaCl-conditioned capa-1 (ok3065) animals in which wild type copies of the capa-1 gene are reintroduced under the control of its promoter sequence ( capa-1p::capa-1::SL2::gfp ). The corresponding mean population position on the gradient, biased random walk and klinotaxis indices are shown on Supplementary Fig. 5b . n ≥ 37 animals per genotype. (i) Chemotactic behavior of NaCl-conditioned animals after ASG-specific RNAi-mediated knockdown of capa-1 , achieved by expressing sense and anti-sense capa-1 sequences under the control of the ASG-specific ops-1 promoter 60 . A strain carrying only the elt-2p ::GFP co-injection marker is used as a control to exclude potential effects of the transgene selection marker. The corresponding mean population position on the gradient, biased random walk and klinotaxis indices are shown on Supplementary Fig. 5c . n ≥ 27 animals per genotype.
    Figure Legend Snippet: CAPA-1 NMU-like neuropeptides signal from ASG chemosensory neurons to promote gustatory learning through NMUR-1 (a) Luminescence-based calcium mobilization assay for measuring GPCR activation. NMUR-1 is expressed in CHO cells that stably co-express the promiscuous human Gα 16 subunit, which couples receptor activation to calcium release from intracellular storage sites. Intracellular calcium levels are monitored using the calcium indicator aequorin. (b) Calcium responses of CHO cells expressing NMUR-1 are shown relative (%) to the highest value (100% activation) after normalization to the total calcium response. Cells transfected with an empty pcDNA3.1 vector are used as a control. Error bars show S.E.M (n ≥ 6). (c) The capa-1/nlp-44 gene encodes a neuropeptide precursor that harbors three predicted peptide sequences (CAPA-1-1, -2, and -3), which are flanked by mono- and dibasic cleaving sites. Black bars indicate the positions of deletions in capa-1/nlp-44 mutant alleles used in this study. (d) Sequence comparison of C. elegans CAPA-1 neuropeptides and NmU neuropeptides from H. sapiens , D. rerio , and D. melanogaster . Conserved C-terminal features are highlighted in black. Residues with similar physicochemical properties are colored gray. (e) Mean position on the gradient through time and (g) chemotactic indices of two independent deletion mutants of the capa-1/nlp-44 neuropeptide precursor gene after mock- and NaCl-conditioning. The corresponding biased random walk and klinotaxis indices are shown on Supplementary Fig. 5a . n ≥ 25 animals per genotype. (f) Co-localization of a bicistronic GFP construct harboring the promoter, genomic DNA and 3’UTR of the capa-1 gene (capa-1p::capa-1::SL2::gfp ) with mCherry expression from the ASG-specific gcy-15 promotor 55 . The asterisk marks fluorescence in the intestine from the elt-2p::gfp co-injection marker. Scale bar, 35 µm. (h) Chemotactic behavior of NaCl-conditioned capa-1 (ok3065) animals in which wild type copies of the capa-1 gene are reintroduced under the control of its promoter sequence ( capa-1p::capa-1::SL2::gfp ). The corresponding mean population position on the gradient, biased random walk and klinotaxis indices are shown on Supplementary Fig. 5b . n ≥ 37 animals per genotype. (i) Chemotactic behavior of NaCl-conditioned animals after ASG-specific RNAi-mediated knockdown of capa-1 , achieved by expressing sense and anti-sense capa-1 sequences under the control of the ASG-specific ops-1 promoter 60 . A strain carrying only the elt-2p ::GFP co-injection marker is used as a control to exclude potential effects of the transgene selection marker. The corresponding mean population position on the gradient, biased random walk and klinotaxis indices are shown on Supplementary Fig. 5c . n ≥ 27 animals per genotype.

    Techniques Used: Calcium Mobilization Assay, Activation Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Sequencing, Construct, Fluorescence, Injection, Marker, Selection

    2) Product Images from "Stathmin is overexpressed and regulated by mutant p53 in oral squamous cell carcinoma"

    Article Title: Stathmin is overexpressed and regulated by mutant p53 in oral squamous cell carcinoma

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0575-4

    Mutant p53 promoted the expression of stathmin. After wtp53, mutp53 (R175H, G245C and R282W) plasmids and vectors were transfected into SCC25 cells, cells transfected with mutp53 grew more aggressively than those with wtp53 ( a ). In SCC25 cells transfected with pcDNA3.1 vector, pcDNA3.1-wtp53 and pcDNA3.1-mutp53 for 48 h, upregulation of stathmin was found in cells transfected with mutp53 but not wtp53 ( b ). Decreased stathmin expression was found accompanied by p53 knockdown for 48 h in two mutant p53 cell lines, HN6 (H179L) and HN13 (V173 L) ( c ). Decreased stathmin mRNA levels were found accompanied by knockdown of p53 for 48 h in HN6 and HN13 cells but not wtp53 HN30 cells ( d , e ). **: P
    Figure Legend Snippet: Mutant p53 promoted the expression of stathmin. After wtp53, mutp53 (R175H, G245C and R282W) plasmids and vectors were transfected into SCC25 cells, cells transfected with mutp53 grew more aggressively than those with wtp53 ( a ). In SCC25 cells transfected with pcDNA3.1 vector, pcDNA3.1-wtp53 and pcDNA3.1-mutp53 for 48 h, upregulation of stathmin was found in cells transfected with mutp53 but not wtp53 ( b ). Decreased stathmin expression was found accompanied by p53 knockdown for 48 h in two mutant p53 cell lines, HN6 (H179L) and HN13 (V173 L) ( c ). Decreased stathmin mRNA levels were found accompanied by knockdown of p53 for 48 h in HN6 and HN13 cells but not wtp53 HN30 cells ( d , e ). **: P

    Techniques Used: Mutagenesis, Expressing, Transfection, Plasmid Preparation

    3) Product Images from "No evidence of autoimmunity to human OX1 or OX2 orexin receptors in Pandemrix-vaccinated narcoleptic children"

    Article Title: No evidence of autoimmunity to human OX1 or OX2 orexin receptors in Pandemrix-vaccinated narcoleptic children

    Journal: Journal of Translational Autoimmunity

    doi: 10.1016/j.jtauto.2020.100055

    Three different vector constructs used to express OX 1 and OX 2 receptor fusion and deletion proteins. (A) OX 1 -GFP and OX 2 -GFP, under a CMV promoter, were expressed in HuH7 cells using a baculovirus-mediated gene delivery method. Panels a and b show the cellular expression pattern of OX 1 -GFP and OX 2 -GFP, respectively, in HuH7 cells. (B) OX 1 -GFP-V5-His and OX 2 -GFP-V5-His, under the CMV promoter in the pcDNA3.1 expression vector, were transiently transfected into HuH7 cells. Panels c and d show the expression pattern of OX 1 -GFP-V5-His and OX 2 -GFP-V5-His in HuH7 cells, respectively. (C) The N-termini of the OX 1 and OX 2 receptors, fused to the myc and His tags, were transiently transfected into HuH7 cells. Panels e and f show the intracellular location and expression pattern of the N-termini of OX 1 and OX 2 receptors, respectively, in HuH7 cells. The calibration bar applies to all images.
    Figure Legend Snippet: Three different vector constructs used to express OX 1 and OX 2 receptor fusion and deletion proteins. (A) OX 1 -GFP and OX 2 -GFP, under a CMV promoter, were expressed in HuH7 cells using a baculovirus-mediated gene delivery method. Panels a and b show the cellular expression pattern of OX 1 -GFP and OX 2 -GFP, respectively, in HuH7 cells. (B) OX 1 -GFP-V5-His and OX 2 -GFP-V5-His, under the CMV promoter in the pcDNA3.1 expression vector, were transiently transfected into HuH7 cells. Panels c and d show the expression pattern of OX 1 -GFP-V5-His and OX 2 -GFP-V5-His in HuH7 cells, respectively. (C) The N-termini of the OX 1 and OX 2 receptors, fused to the myc and His tags, were transiently transfected into HuH7 cells. Panels e and f show the intracellular location and expression pattern of the N-termini of OX 1 and OX 2 receptors, respectively, in HuH7 cells. The calibration bar applies to all images.

    Techniques Used: Plasmid Preparation, Construct, Expressing, Transfection

    4) Product Images from "Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer"

    Article Title: Role of SMC1 in Overcoming Drug Resistance in Triple Negative Breast Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064338

    Effect of SMC1 on cellular Viability and Anchorage Independent Cell Growth. The MDA-MB-231 cells were transfected with the eukaryotic expression vector alone (pcDNA3.1) and SMC1/pcDNA3.1, using Lipofectamine 2000 transfection reagent (Invitrogen). Transfection of scrambled and SMC1 siRNA was performed with Lipofectamine RNAiMax kit (Invitrogen) following manufactures instructions. Total RNA was purified using Trizol reagent and quantified using a nano-drop spectrophotometer (Thermo Scientific). Expression of SMC1 mRNA was evaluated by RT-PCR analysis using gene specific primers [nt 307–326 bp (upstream primer) and nt 730–750 bp (downstream primer)] and β-actin was used as a control. Equal amount of DNA was loaded on 1% agarose gel; lane 1, pcDNA3.1 (control vector); lane 2, pcDNA3.1/SMC1; lane 3, scrambled siRNA; and lane 4 SMC1 siRNA to check for the overexpression and silencing of SMC1( Panel A ). For SMC1-liposomes, SMC1 was purified by DNP-SG affinity chromatography and reconstituted into liposomes using our established procedure [36] , [38] . Viability of colonies was determined by colony forming assay, performed in untreated, control-liposomes, SMC1-liposomes, scrambled and SMC1 siRNA, pcDNA3.1 and SMC1/pcDNA3.1 transfected MDA-MB-231 cells (0.1×10 6 cells/500 µl in triplicates). Aliquots of 50 and 100 µl (in triplicates) were taken in 60 mm size petri-dishes, separately, in a total volume of 4 ml with medium. After 10 days, cells were stained with 0.5% methylene blue for 30 min and colonies were counted using Innotech Alpha Imager. The results shown are normalized to control untreated cells. Values are means ± S.D. of three separate experiments ( Panel B ). The effect of SMC1 overexpression (SMC1 transfected) and suppression (SMC1 siRNA) was also checked on the cell apoptosis by using TUNEL apoptosis kit (Promega). Briefly, approximately 0.1×10 6 MDA-MB-231 cells were grown on the cover slips in 12 well plate and transfected with SMC1/pcDNA3.1 and SMC1 siRNA as described in method section. TUNEL apoptosis assay was performed using the Promega Apoptosis Detection Kit according to manufactures instructions. The slides were analyzed by fluorescence microscope (Olympus IX81 automated Inverted) using a standard fluorescein filter set to view the green fluorescence at 520 nm, and blue fluorescence at > 340 nm. Photographs taken at identical exposure at 20× magnification are presented. Apoptotic cells showed green fluorescence and characteristic cell shrinkage ( Panel C ). MDA-MB-231 cells transfected with scrambled shRNA and SMC1 shRNA were also tested for the anchorage-independent growth on soft agar as described in the methods section and colonies were counted after 21 days and plotted ( Panel D ).
    Figure Legend Snippet: Effect of SMC1 on cellular Viability and Anchorage Independent Cell Growth. The MDA-MB-231 cells were transfected with the eukaryotic expression vector alone (pcDNA3.1) and SMC1/pcDNA3.1, using Lipofectamine 2000 transfection reagent (Invitrogen). Transfection of scrambled and SMC1 siRNA was performed with Lipofectamine RNAiMax kit (Invitrogen) following manufactures instructions. Total RNA was purified using Trizol reagent and quantified using a nano-drop spectrophotometer (Thermo Scientific). Expression of SMC1 mRNA was evaluated by RT-PCR analysis using gene specific primers [nt 307–326 bp (upstream primer) and nt 730–750 bp (downstream primer)] and β-actin was used as a control. Equal amount of DNA was loaded on 1% agarose gel; lane 1, pcDNA3.1 (control vector); lane 2, pcDNA3.1/SMC1; lane 3, scrambled siRNA; and lane 4 SMC1 siRNA to check for the overexpression and silencing of SMC1( Panel A ). For SMC1-liposomes, SMC1 was purified by DNP-SG affinity chromatography and reconstituted into liposomes using our established procedure [36] , [38] . Viability of colonies was determined by colony forming assay, performed in untreated, control-liposomes, SMC1-liposomes, scrambled and SMC1 siRNA, pcDNA3.1 and SMC1/pcDNA3.1 transfected MDA-MB-231 cells (0.1×10 6 cells/500 µl in triplicates). Aliquots of 50 and 100 µl (in triplicates) were taken in 60 mm size petri-dishes, separately, in a total volume of 4 ml with medium. After 10 days, cells were stained with 0.5% methylene blue for 30 min and colonies were counted using Innotech Alpha Imager. The results shown are normalized to control untreated cells. Values are means ± S.D. of three separate experiments ( Panel B ). The effect of SMC1 overexpression (SMC1 transfected) and suppression (SMC1 siRNA) was also checked on the cell apoptosis by using TUNEL apoptosis kit (Promega). Briefly, approximately 0.1×10 6 MDA-MB-231 cells were grown on the cover slips in 12 well plate and transfected with SMC1/pcDNA3.1 and SMC1 siRNA as described in method section. TUNEL apoptosis assay was performed using the Promega Apoptosis Detection Kit according to manufactures instructions. The slides were analyzed by fluorescence microscope (Olympus IX81 automated Inverted) using a standard fluorescein filter set to view the green fluorescence at 520 nm, and blue fluorescence at > 340 nm. Photographs taken at identical exposure at 20× magnification are presented. Apoptotic cells showed green fluorescence and characteristic cell shrinkage ( Panel C ). MDA-MB-231 cells transfected with scrambled shRNA and SMC1 shRNA were also tested for the anchorage-independent growth on soft agar as described in the methods section and colonies were counted after 21 days and plotted ( Panel D ).

    Techniques Used: Multiple Displacement Amplification, Transfection, Expressing, Plasmid Preparation, Purification, Spectrophotometry, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Over Expression, Affinity Chromatography, Staining, TUNEL Assay, Apoptosis Assay, Fluorescence, Microscopy, shRNA

    5) Product Images from "A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation"

    Article Title: A novel human truncated IL12rβ1-Fc fusion protein ameliorates experimental autoimmune encephalomyelitis via specific binding of p40 to inhibit Th1 and Th17 cell differentiation

    Journal: Oncotarget

    doi:

    Construction, expression, and purification of tIL12rβ1/Fc A. Plasmid map of the eukaryotic expression plasmid containing human truncated IL-12rβ1 receptor (pcDNA3.1(+)-tIL12rβ1/Fc). The gene sequence encoding tIL12rβ1/Fc was inserted into pcDNA3.1(+) vector at the corresponding restriction sites Hind III and Xho I. B. PCR products of tIL12rβ1/Fc recombinant gene. Lane 1, tIL12rβ1/Fc gene sequence containing the signal peptide. Lane 2, IgG1 Fc fragment. Lane 3, recombinant tIL12rβ1/Fc fused gene product using overlap PCR. M, DNA molecular weight markers, bp. C. Correctly constructed plasmid was verified by digestion at the Hind III and Xho I sites. Lane 1, pcDNA3.1(+)-tIL12rβ1/Fc after digestion. Lane 2, pcDNA3.1(+) -tIL12rβ1/Fc before digestion. M, DNA molecular weight markers, bp. D. Plasmids were linearized for cell transfection using Pvu I. Lane 1 and 2, linearized pcDNA3.1(+)-tIL12rβ1/Fc. M, DNA molecular weight markers, bp. E. Total RNA of the two strains were identified by RT-PCR with primers F1 and F4. F. SDS-PAGE analysis of the purified tIL12rβ1/Fc fusion protein using Protein A column. Lane 1, purified tIL12rβ1/Fc fusion protein at reduced state. Lane 2, purified tIL12rβ1/Fc fusion protein at non-reduced state. M, protein molecular weight markers, KDa. G. Western blot analysis of tIL12rβ1/Fc using mAbs against human IL12rβ1. Lane 1, purified tIL12rβ1/Fc fusion protein at reduced state. Lane 2, purified tIL12rβ1/Fc fusion protein at non-reduced state. M, protein molecular weight markers, KDa.
    Figure Legend Snippet: Construction, expression, and purification of tIL12rβ1/Fc A. Plasmid map of the eukaryotic expression plasmid containing human truncated IL-12rβ1 receptor (pcDNA3.1(+)-tIL12rβ1/Fc). The gene sequence encoding tIL12rβ1/Fc was inserted into pcDNA3.1(+) vector at the corresponding restriction sites Hind III and Xho I. B. PCR products of tIL12rβ1/Fc recombinant gene. Lane 1, tIL12rβ1/Fc gene sequence containing the signal peptide. Lane 2, IgG1 Fc fragment. Lane 3, recombinant tIL12rβ1/Fc fused gene product using overlap PCR. M, DNA molecular weight markers, bp. C. Correctly constructed plasmid was verified by digestion at the Hind III and Xho I sites. Lane 1, pcDNA3.1(+)-tIL12rβ1/Fc after digestion. Lane 2, pcDNA3.1(+) -tIL12rβ1/Fc before digestion. M, DNA molecular weight markers, bp. D. Plasmids were linearized for cell transfection using Pvu I. Lane 1 and 2, linearized pcDNA3.1(+)-tIL12rβ1/Fc. M, DNA molecular weight markers, bp. E. Total RNA of the two strains were identified by RT-PCR with primers F1 and F4. F. SDS-PAGE analysis of the purified tIL12rβ1/Fc fusion protein using Protein A column. Lane 1, purified tIL12rβ1/Fc fusion protein at reduced state. Lane 2, purified tIL12rβ1/Fc fusion protein at non-reduced state. M, protein molecular weight markers, KDa. G. Western blot analysis of tIL12rβ1/Fc using mAbs against human IL12rβ1. Lane 1, purified tIL12rβ1/Fc fusion protein at reduced state. Lane 2, purified tIL12rβ1/Fc fusion protein at non-reduced state. M, protein molecular weight markers, KDa.

    Techniques Used: Expressing, Purification, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Recombinant, Molecular Weight, Construct, Transfection, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot

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    Article Snippet: .. The ORF was subcloned into pcDNA3.1(+) (Invitrogen) by using specific restriction sites HindIII/XhoI (CNT3) and NotI/XhoI (CD73) for expression in COS-7 cells. ..

    Article Title: Dysadherin, a cancer-associated cell membrane glycoprotein, down-regulates E-cadherin and promotes metastasis
    Article Snippet: .. A cDNA fragment of dysadherin containing the entire ORF without a stop codon was amplified by using L3 cDNA as a template by PCR and subcloned into a mammalian expression vector pcDNA3 (Invitrogen) with a C-terminal herpes simplex virus (HSV) tag sequence (Novagen). .. This construct, L3 HSV/pcDNA3, was sequenced to exclude mismatches.

    Polymerase Chain Reaction:

    Article Title: A viral gene that activates lytic cycle expression of Kaposi's sarcoma-associated herpesvirus
    Article Snippet: .. These PCR fragments were cloned into the pcDNA3.1 expression vector (Invitrogen) and designated as KSHV/gRta. .. Primer A and primer B were used to generate PCR products from total BC-1 DNA in a separate reaction.

    Article Title: Dysadherin, a cancer-associated cell membrane glycoprotein, down-regulates E-cadherin and promotes metastasis
    Article Snippet: .. A cDNA fragment of dysadherin containing the entire ORF without a stop codon was amplified by using L3 cDNA as a template by PCR and subcloned into a mammalian expression vector pcDNA3 (Invitrogen) with a C-terminal herpes simplex virus (HSV) tag sequence (Novagen). .. This construct, L3 HSV/pcDNA3, was sequenced to exclude mismatches.

    Over Expression:

    Article Title: A novel long non-coding RNA-PRLB acts as a tumor promoter through regulating miR-4766-5p/SIRT1 axis in breast cancer
    Article Snippet: .. Psmid construction and transfection For overexpression of lncRNA-PRLB, lncRNA-PRLB cDNA was cloned into the multiple cloning site of the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). .. Both the expression plasmid vector and the empty vector were used to transfect breast cancer cells using Lipofectamine 2000 (Invitrogen, MA, USA) to establish lncRNA-PRLB overexpression and control cell lines, screened with puromycin (2 μg/ml) for 4 weeks.

    Plasmid Preparation:

    Article Title: Kilham Polyomavirus: Activation of Gene Expression and DNA Replication in Mouse Fibroblast Cells by an Enhancer Substitution
    Article Snippet: .. KV and PyV large T antigens were expressed by inserting the coding sequence of the protein into the vector pcDNA3 (Invitrogen), which contains the cytomegalovirus immediate-early promoter. ..

    Article Title: A novel long non-coding RNA-PRLB acts as a tumor promoter through regulating miR-4766-5p/SIRT1 axis in breast cancer
    Article Snippet: .. Psmid construction and transfection For overexpression of lncRNA-PRLB, lncRNA-PRLB cDNA was cloned into the multiple cloning site of the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). .. Both the expression plasmid vector and the empty vector were used to transfect breast cancer cells using Lipofectamine 2000 (Invitrogen, MA, USA) to establish lncRNA-PRLB overexpression and control cell lines, screened with puromycin (2 μg/ml) for 4 weeks.

    Article Title: The Gfi-1B Proto-Oncoprotein Represses p21WAF1 and Inhibits Myeloid Cell Differentiation
    Article Snippet: .. To generate pCMV5/Gfi-1B, the 1.7-kb full-length Gfi-1B cDNA was released from pBluescript(SK−) by double digestion with Eco RI and Sal I and then subcloned between Eco RI and Sal I in the CMV5 expression vector ( ) or between Eco RI and Xho I in the expression vector pcDNA3 (Invitrogen). .. To generate the VP16/Gfi-1B chimera, the sequence encoding the acidic activation domain of VP16 , including the Kozak consensus, was amplified by PCR.

    Article Title: A viral gene that activates lytic cycle expression of Kaposi's sarcoma-associated herpesvirus
    Article Snippet: .. These PCR fragments were cloned into the pcDNA3.1 expression vector (Invitrogen) and designated as KSHV/gRta. .. Primer A and primer B were used to generate PCR products from total BC-1 DNA in a separate reaction.

    Article Title: The chimeric gene CHRFAM7A, a partial duplication of the CHRNA7 gene, is a dominant negative regulator of α7*nAChR function
    Article Snippet: .. After TA cloning, the fragment was moved to pcDNA3.1 (Invitrogen, Carlsbad, CA), a mammalian expression vector. .. The 2bp deletion in exon 6 of CHRFAM7A was introduced into pcDNA3.1 CHRFAM7A by PCR with the QuikChange II XL Site-Directed Mutagenesis Kit, according to the manufacturer's protocols (Stratagene, La Jolly, CA).

    Article Title: MicroRNA-338-3p suppresses cell proliferation and induces apoptosis of non-small-cell lung cancer by targeting sphingosine kinase 2
    Article Snippet: .. Plasmid construction and cell transfection SphK2 coding sequences lacking the 3′-UTR were cloned into the pcDNA3.1 vector (Invitrogen) to generate the pcDNA3.1-SphK2 expression vector. ..

    Article Title: Dysadherin, a cancer-associated cell membrane glycoprotein, down-regulates E-cadherin and promotes metastasis
    Article Snippet: .. A cDNA fragment of dysadherin containing the entire ORF without a stop codon was amplified by using L3 cDNA as a template by PCR and subcloned into a mammalian expression vector pcDNA3 (Invitrogen) with a C-terminal herpes simplex virus (HSV) tag sequence (Novagen). .. This construct, L3 HSV/pcDNA3, was sequenced to exclude mismatches.

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    Thermo Fisher eukaryotic expression vector pcdna3 1
    Three different vector constructs used to express OX 1 and OX 2 receptor fusion and deletion proteins. (A) OX 1 -GFP and OX 2 -GFP, under a CMV promoter, were expressed in HuH7 cells using a baculovirus-mediated gene delivery method. Panels a and b show the cellular expression pattern of OX 1 -GFP and OX 2 -GFP, respectively, in HuH7 cells. (B) OX 1 -GFP-V5-His and OX 2 -GFP-V5-His, under the CMV promoter in the <t>pcDNA3.1</t> expression vector, were transiently transfected into HuH7 cells. Panels c and d show the expression pattern of OX 1 -GFP-V5-His and OX 2 -GFP-V5-His in HuH7 cells, respectively. (C) The N-termini of the OX 1 and OX 2 receptors, fused to the myc and His tags, were transiently transfected into HuH7 cells. Panels e and f show the intracellular location and expression pattern of the N-termini of OX 1 and OX 2 receptors, respectively, in HuH7 cells. The calibration bar applies to all images.
    Eukaryotic Expression Vector Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Three different vector constructs used to express OX 1 and OX 2 receptor fusion and deletion proteins. (A) OX 1 -GFP and OX 2 -GFP, under a CMV promoter, were expressed in HuH7 cells using a baculovirus-mediated gene delivery method. Panels a and b show the cellular expression pattern of OX 1 -GFP and OX 2 -GFP, respectively, in HuH7 cells. (B) OX 1 -GFP-V5-His and OX 2 -GFP-V5-His, under the CMV promoter in the pcDNA3.1 expression vector, were transiently transfected into HuH7 cells. Panels c and d show the expression pattern of OX 1 -GFP-V5-His and OX 2 -GFP-V5-His in HuH7 cells, respectively. (C) The N-termini of the OX 1 and OX 2 receptors, fused to the myc and His tags, were transiently transfected into HuH7 cells. Panels e and f show the intracellular location and expression pattern of the N-termini of OX 1 and OX 2 receptors, respectively, in HuH7 cells. The calibration bar applies to all images.

    Journal: Journal of Translational Autoimmunity

    Article Title: No evidence of autoimmunity to human OX1 or OX2 orexin receptors in Pandemrix-vaccinated narcoleptic children

    doi: 10.1016/j.jtauto.2020.100055

    Figure Lengend Snippet: Three different vector constructs used to express OX 1 and OX 2 receptor fusion and deletion proteins. (A) OX 1 -GFP and OX 2 -GFP, under a CMV promoter, were expressed in HuH7 cells using a baculovirus-mediated gene delivery method. Panels a and b show the cellular expression pattern of OX 1 -GFP and OX 2 -GFP, respectively, in HuH7 cells. (B) OX 1 -GFP-V5-His and OX 2 -GFP-V5-His, under the CMV promoter in the pcDNA3.1 expression vector, were transiently transfected into HuH7 cells. Panels c and d show the expression pattern of OX 1 -GFP-V5-His and OX 2 -GFP-V5-His in HuH7 cells, respectively. (C) The N-termini of the OX 1 and OX 2 receptors, fused to the myc and His tags, were transiently transfected into HuH7 cells. Panels e and f show the intracellular location and expression pattern of the N-termini of OX 1 and OX 2 receptors, respectively, in HuH7 cells. The calibration bar applies to all images.

    Article Snippet: Influenza A/WSN/33 NP (accession number: CY034135.1 ) gene was modified by PCR to create 5′ and 3′ BamHI sites and an N-terminal Kozak consensus sequence for its further cloning into the eukaryotic expression vector pcDNA3.1 (+) (Thermo Fisher Scientific Inc.).

    Techniques: Plasmid Preparation, Construct, Expressing, Transfection

    POSTN immunostaining in hCh-1 cells. To detect POSTN protein in hCh-1 nontransfected cells (control, n = 3), vehicle-transfected cells (vehicle, n = 3), and cells transfected with pcDNA3.1-hPOSTN-001 ( hPOSTN-001 , n = 3), cells were subjected to immunofluorescence. Phalloidin (red-orange) was used to visualize F-actin, DAPI (blue) staining showed nuclei, and POSTN was shown by green staining. Magnification value, ×400. Quantification showed that control and hPOSTN-001 cells had significantly higher levels of POSTN signals than vehicle cells. These data reflected the same pattern as shown by real-time PCR. Similar lowercase letters ( a , b ) in each graph represent statistical significance from each other at P

    Journal: The FASEB Journal

    Article Title: Distinct expression pattern of periostin splice variants in chondrocytes and ligament progenitor cells

    doi: 10.1096/fj.201802281R

    Figure Lengend Snippet: POSTN immunostaining in hCh-1 cells. To detect POSTN protein in hCh-1 nontransfected cells (control, n = 3), vehicle-transfected cells (vehicle, n = 3), and cells transfected with pcDNA3.1-hPOSTN-001 ( hPOSTN-001 , n = 3), cells were subjected to immunofluorescence. Phalloidin (red-orange) was used to visualize F-actin, DAPI (blue) staining showed nuclei, and POSTN was shown by green staining. Magnification value, ×400. Quantification showed that control and hPOSTN-001 cells had significantly higher levels of POSTN signals than vehicle cells. These data reflected the same pattern as shown by real-time PCR. Similar lowercase letters ( a , b ) in each graph represent statistical significance from each other at P

    Article Snippet: The insert was chemically synthesized and inserted into Bam HI and Eco RI (Thermo Fisher Scientific) sites of eukaryotic expression vector pcDNA3.1(+) plasmid including a neomycin-resistant gene (Thermo Fisher Scientific).

    Techniques: Immunostaining, Transfection, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction

    Expression pattern of POSTN and its splice variants in hCh-1 cells stably transfected clones. To determine whether POSTN and its splice variants are differentially expressed, we compared the expression profile of nontransfected hCh-1 cells (control, n = 3), vehicle cells (vehicle, n = 3), and cells transfected with pcDNA3.1–hPOSTN-001 ( hPOSTN-001 , n = 3). We observed that POSTN mRNA was significantly higher in control compared with vehicle cells. POSTN isoform 1 was significantly higher on hPOSTN-001 cells compared with control and vehicle cells. Lowercase letters ( a ) in each graph represent statistical significance from each other at P

    Journal: The FASEB Journal

    Article Title: Distinct expression pattern of periostin splice variants in chondrocytes and ligament progenitor cells

    doi: 10.1096/fj.201802281R

    Figure Lengend Snippet: Expression pattern of POSTN and its splice variants in hCh-1 cells stably transfected clones. To determine whether POSTN and its splice variants are differentially expressed, we compared the expression profile of nontransfected hCh-1 cells (control, n = 3), vehicle cells (vehicle, n = 3), and cells transfected with pcDNA3.1–hPOSTN-001 ( hPOSTN-001 , n = 3). We observed that POSTN mRNA was significantly higher in control compared with vehicle cells. POSTN isoform 1 was significantly higher on hPOSTN-001 cells compared with control and vehicle cells. Lowercase letters ( a ) in each graph represent statistical significance from each other at P

    Article Snippet: The insert was chemically synthesized and inserted into Bam HI and Eco RI (Thermo Fisher Scientific) sites of eukaryotic expression vector pcDNA3.1(+) plasmid including a neomycin-resistant gene (Thermo Fisher Scientific).

    Techniques: Expressing, Stable Transfection, Transfection, Clone Assay

    MiR-101-3p inhibits PI3K-AKT-mTOR pathway via STC1 . Goat granulosa cells are transfected (a) miR-101-3p mimics (miR-101-3p-mi) or mimics NC (NC-mi), miR-101-3p inhibitors (miR-101-3p-in) or inhibitors NC (NC-in), (b) siRNA-STC1 (si-STC1) or NC, pcDNA3.1-STC1 or pcDNA3.1 (control), (c) miR-101-3p-mi or NC-mi or co-transfected miR-101–3p-mi with pcDNA3.1-STC1 for 48 h. Cytosolic protein and related phosphorylation levels for PI3K, AKT, mTOR and PTEN are analysed using Western blot. β-actin is used as an internal control. Values are expressed as mean ± SD of n = 3. * P

    Journal: Journal of Animal Science and Biotechnology

    Article Title: Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1

    doi: 10.1186/s40104-020-00506-6

    Figure Lengend Snippet: MiR-101-3p inhibits PI3K-AKT-mTOR pathway via STC1 . Goat granulosa cells are transfected (a) miR-101-3p mimics (miR-101-3p-mi) or mimics NC (NC-mi), miR-101-3p inhibitors (miR-101-3p-in) or inhibitors NC (NC-in), (b) siRNA-STC1 (si-STC1) or NC, pcDNA3.1-STC1 or pcDNA3.1 (control), (c) miR-101-3p-mi or NC-mi or co-transfected miR-101–3p-mi with pcDNA3.1-STC1 for 48 h. Cytosolic protein and related phosphorylation levels for PI3K, AKT, mTOR and PTEN are analysed using Western blot. β-actin is used as an internal control. Values are expressed as mean ± SD of n = 3. * P

    Article Snippet: Afterwards, STC1 overexpression plasmids were constructed using the eukaryotic expression pcDNA3.1(+) vector (Thermo Fisher, Shanghai, China) between Hind III and Xho I sites.

    Techniques: Transfection, Western Blot

    MiR-101-3p inhibits granulosa cell proliferation via STC1 . Goat granulosa cells are transfected with miR-101-3p mimics (miR-101-3p-mi) or mimics NC (NC-mi), miR-101-3p inhibitors (miR-101-3p-in) or inhibitors NC (NC-in), siRNA-STC1 (si-STC1) or NC, pcDNA3.1-STC1 or pcDNA3.1 (control). a Cell viability is standardized by relative absorbance (OD values at 450 nm) and determined using CCK8 assay at 12, 24, 48 and 72 h time points. After 24 h transfection, (b , c) granulosa cells in the S-phase are stained with EdU in red, whereas cell nuclei are dyed with DAPI in blue. The ratio of red and blue dyed cell numbers represents the percentage of EdU positive cells. Cell cycle distribution of granulosa cells is measured using flow cytometry. d The percentage of EdU positive cells or cell cycle distribution is detected using above-mentioned methods in granulosa cells transfected with miR-101-3p-mi or NC-mi or co-transfected miR-101-3p-mi with pcDNA3.1-STC1 for 24 h. Values are expressed as mean ± SD of n = 3. * P

    Journal: Journal of Animal Science and Biotechnology

    Article Title: Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1

    doi: 10.1186/s40104-020-00506-6

    Figure Lengend Snippet: MiR-101-3p inhibits granulosa cell proliferation via STC1 . Goat granulosa cells are transfected with miR-101-3p mimics (miR-101-3p-mi) or mimics NC (NC-mi), miR-101-3p inhibitors (miR-101-3p-in) or inhibitors NC (NC-in), siRNA-STC1 (si-STC1) or NC, pcDNA3.1-STC1 or pcDNA3.1 (control). a Cell viability is standardized by relative absorbance (OD values at 450 nm) and determined using CCK8 assay at 12, 24, 48 and 72 h time points. After 24 h transfection, (b , c) granulosa cells in the S-phase are stained with EdU in red, whereas cell nuclei are dyed with DAPI in blue. The ratio of red and blue dyed cell numbers represents the percentage of EdU positive cells. Cell cycle distribution of granulosa cells is measured using flow cytometry. d The percentage of EdU positive cells or cell cycle distribution is detected using above-mentioned methods in granulosa cells transfected with miR-101-3p-mi or NC-mi or co-transfected miR-101-3p-mi with pcDNA3.1-STC1 for 24 h. Values are expressed as mean ± SD of n = 3. * P

    Article Snippet: Afterwards, STC1 overexpression plasmids were constructed using the eukaryotic expression pcDNA3.1(+) vector (Thermo Fisher, Shanghai, China) between Hind III and Xho I sites.

    Techniques: Transfection, CCK-8 Assay, Staining, Flow Cytometry

    MiR-101-3p regulates the apoptotic genes in granulosa cells via STC1 . Goat granulosa cells are transfected with miR-101-3p mimics (miR-101-3p-mi) or mimics NC (NC-mi), miR-101-3p inhibitors (miR-101-3p-in) or inhibitors NC (NC-in). a The mRNA expressions of apoptotic genes ( Bcl-2, Bax and p53 ) are detected using RT-qPCR after 24 h transfection. b The protein expressions of apoptotic genes (Caspase 3, Bcl-2, Bax and p53) are detected using Western blot after 48 h transfection. Caspase 3, Bcl-2, Bax and p53 protein levels are detected in granulosa cells transfected with (d) siRNA-STC1 (si-STC1) or NC, pcDNA3.1-STC1 or pcDNA3.1 (control), (e) miR-101-3p-mi or NC-mi or co-transfected miR-101-3p-mi with pcDNA3.1-STC1 for 48 h using Western blot. (c, f) Histograms show the ratio of Bcl-2/Bax. β-actin is used as an internal control. Values are expressed as mean ± SD of n = 3. * P

    Journal: Journal of Animal Science and Biotechnology

    Article Title: Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1

    doi: 10.1186/s40104-020-00506-6

    Figure Lengend Snippet: MiR-101-3p regulates the apoptotic genes in granulosa cells via STC1 . Goat granulosa cells are transfected with miR-101-3p mimics (miR-101-3p-mi) or mimics NC (NC-mi), miR-101-3p inhibitors (miR-101-3p-in) or inhibitors NC (NC-in). a The mRNA expressions of apoptotic genes ( Bcl-2, Bax and p53 ) are detected using RT-qPCR after 24 h transfection. b The protein expressions of apoptotic genes (Caspase 3, Bcl-2, Bax and p53) are detected using Western blot after 48 h transfection. Caspase 3, Bcl-2, Bax and p53 protein levels are detected in granulosa cells transfected with (d) siRNA-STC1 (si-STC1) or NC, pcDNA3.1-STC1 or pcDNA3.1 (control), (e) miR-101-3p-mi or NC-mi or co-transfected miR-101-3p-mi with pcDNA3.1-STC1 for 48 h using Western blot. (c, f) Histograms show the ratio of Bcl-2/Bax. β-actin is used as an internal control. Values are expressed as mean ± SD of n = 3. * P

    Article Snippet: Afterwards, STC1 overexpression plasmids were constructed using the eukaryotic expression pcDNA3.1(+) vector (Thermo Fisher, Shanghai, China) between Hind III and Xho I sites.

    Techniques: Transfection, Quantitative RT-PCR, Western Blot

    MiR-101-3p inhibits the expressions of cell proliferation-related genes via STC1 . Goat granulosa cells are transfected with miR-101-3p mimics (miR-101-3p-mi) or mimics NC (NC-mi), miR-101-3p inhibitors (miR-101-3p-in) or inhibitors NC (NC-in). The (a) mRNA or (b) protein expressions of cell proliferation-related genes ( CDK4, CCND1, CCNE1 and PCNA ) are detected using RT-qPCR or Western blot after 24 h or 48 h transfection. Western blot measures the protein expressions of CDK4, CCND1, CCNE1 and PCNA in granulosa cells transfected with (c) siRNA-STC1 (si-STC1) or NC, pcDNA3.1-STC1 or pcDNA3.1 (control) or (d) miR-101-3p-mi or NC-mi or co-transfected miR-101-3p-mi with pcDNA3.1-STC1 for 48 h. β-actin is used as an internal control. Values are expressed as mean ± SD of n = 3. * P

    Journal: Journal of Animal Science and Biotechnology

    Article Title: Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1

    doi: 10.1186/s40104-020-00506-6

    Figure Lengend Snippet: MiR-101-3p inhibits the expressions of cell proliferation-related genes via STC1 . Goat granulosa cells are transfected with miR-101-3p mimics (miR-101-3p-mi) or mimics NC (NC-mi), miR-101-3p inhibitors (miR-101-3p-in) or inhibitors NC (NC-in). The (a) mRNA or (b) protein expressions of cell proliferation-related genes ( CDK4, CCND1, CCNE1 and PCNA ) are detected using RT-qPCR or Western blot after 24 h or 48 h transfection. Western blot measures the protein expressions of CDK4, CCND1, CCNE1 and PCNA in granulosa cells transfected with (c) siRNA-STC1 (si-STC1) or NC, pcDNA3.1-STC1 or pcDNA3.1 (control) or (d) miR-101-3p-mi or NC-mi or co-transfected miR-101-3p-mi with pcDNA3.1-STC1 for 48 h. β-actin is used as an internal control. Values are expressed as mean ± SD of n = 3. * P

    Article Snippet: Afterwards, STC1 overexpression plasmids were constructed using the eukaryotic expression pcDNA3.1(+) vector (Thermo Fisher, Shanghai, China) between Hind III and Xho I sites.

    Techniques: Transfection, Quantitative RT-PCR, Western Blot

    MiR-101-3p promotes granulosa cell apoptosis via STC1 . Goat granulosa cells are transfected with (a) miR-101-3p mimics (miR-101-3p-mi) or mimics NC (NC-mi), miR-101-3p inhibitors (miR-101-3p-in) or inhibitors NC (NC-in), (b) siRNA-STC1 (si-STC1) or NC, pcDNA3.1-STC1 or pcDNA3.1 (control), (c) miR-101-3p-mi or NC-mi or co-transfected miR-101-3p-mi with pcDNA3.1-STC1 for 24 h. The apoptotic rates of granulosa cells are detected by flow cytometry. Histograms show the apoptotic cell distribution and percentage. Q1 means death cells, Q2 means late-state apoptotic cells, Q3 means early-state apoptotic cells and Q4 means viable non-apoptotic cells. Values are expressed as mean ± SD of n = 3. * P

    Journal: Journal of Animal Science and Biotechnology

    Article Title: Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1

    doi: 10.1186/s40104-020-00506-6

    Figure Lengend Snippet: MiR-101-3p promotes granulosa cell apoptosis via STC1 . Goat granulosa cells are transfected with (a) miR-101-3p mimics (miR-101-3p-mi) or mimics NC (NC-mi), miR-101-3p inhibitors (miR-101-3p-in) or inhibitors NC (NC-in), (b) siRNA-STC1 (si-STC1) or NC, pcDNA3.1-STC1 or pcDNA3.1 (control), (c) miR-101-3p-mi or NC-mi or co-transfected miR-101-3p-mi with pcDNA3.1-STC1 for 24 h. The apoptotic rates of granulosa cells are detected by flow cytometry. Histograms show the apoptotic cell distribution and percentage. Q1 means death cells, Q2 means late-state apoptotic cells, Q3 means early-state apoptotic cells and Q4 means viable non-apoptotic cells. Values are expressed as mean ± SD of n = 3. * P

    Article Snippet: Afterwards, STC1 overexpression plasmids were constructed using the eukaryotic expression pcDNA3.1(+) vector (Thermo Fisher, Shanghai, China) between Hind III and Xho I sites.

    Techniques: Transfection, Flow Cytometry

    MiR-101-3p promotes steroid hormone synthesis via STC1 . ELISA kits measure oestrogen (E2) and progesterone (P4) secretions in 50 μL cell-free supernatants in goat granulosa cells transfected with (a) siRNA-STC1 (si-STC1) or NC, pcDNA3.1-STC1 or pcDNA3.1 (control) or (b) miR-101-3p mimics (miR-101-3p-mi), mimics NC (NC-mi) or co-transfected miR-101-3p-mi with pcDNA3.1-STC1 for 24 h. The protein expressions of steroid hormone synthesis-associated vital genes STAR, CYP11A1, CYP19A1 and 3β-HSD are quantified using Western blot in goat granulosa cells transfected with (c) si-STC1 or NC, pcDNA3.1-STC1 or pcDNA3.1 (control) or (d) miR-101-3p-mi, NC-mi or co-transfected miR-101-3p-mi with pcDNA3.1-STC1 after 48 h transfection. β-actin is used as an internal control. Values are expressed as mean ± SD of n = 3. * P

    Journal: Journal of Animal Science and Biotechnology

    Article Title: Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1

    doi: 10.1186/s40104-020-00506-6

    Figure Lengend Snippet: MiR-101-3p promotes steroid hormone synthesis via STC1 . ELISA kits measure oestrogen (E2) and progesterone (P4) secretions in 50 μL cell-free supernatants in goat granulosa cells transfected with (a) siRNA-STC1 (si-STC1) or NC, pcDNA3.1-STC1 or pcDNA3.1 (control) or (b) miR-101-3p mimics (miR-101-3p-mi), mimics NC (NC-mi) or co-transfected miR-101-3p-mi with pcDNA3.1-STC1 for 24 h. The protein expressions of steroid hormone synthesis-associated vital genes STAR, CYP11A1, CYP19A1 and 3β-HSD are quantified using Western blot in goat granulosa cells transfected with (c) si-STC1 or NC, pcDNA3.1-STC1 or pcDNA3.1 (control) or (d) miR-101-3p-mi, NC-mi or co-transfected miR-101-3p-mi with pcDNA3.1-STC1 after 48 h transfection. β-actin is used as an internal control. Values are expressed as mean ± SD of n = 3. * P

    Article Snippet: Afterwards, STC1 overexpression plasmids were constructed using the eukaryotic expression pcDNA3.1(+) vector (Thermo Fisher, Shanghai, China) between Hind III and Xho I sites.

    Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Western Blot