pspl3 vector Search Results


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  • 74
    Addgene inc pspl3 vector
    Pspl3 Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 74/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pspl3 vector/product/Addgene inc
    Average 74 stars, based on 1 article reviews
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    79
    Thermo Fisher pspl3 vector
    SNP g.18174 A > G induces aberrant NCF4 - TV splicing. (A) Schematic representation of the NCF4 mini-genes used in the functional splicing assay. The wild-type and mutant fragments contained 125 bp of intron 9 and 53 bp of exon 10; fragments harboring the A or G allele were separately cloned into the Eco RI and Xho I cloning sites of the <t>pSPL3</t> vector. Two mini-gene expression vectors were transiently transfected into 293T cells. (B) RT-PCR analysis of the NCF4 spliced transcripts on a 2% agarose gel. RT-PCR products were amplified from the total RNA of 293T cells transfected with the wild-type and mutant (g.18174 A > G) NCF4 mini-gene constructs. The size of the RT-PCR product (441 bp) corresponded to the amplified portion of intron 9 (77 bp), the retained portion of intron 9 (48 bp), the amplification of exon 10 (53 bp), and the pSPL3 control plasmid (263 bp). The size of the RT-PCR product (393 bp) corresponded to the amplified portion of intron 9 (77 bp), the amplification of exon 10 (53 bp), and the pSPL3 control plasmid (263 bp). (C) Electrophoresis of RT-PCR products showing the presence and abundance of NCF4 - TV transcript in bovine mammary samples with three NCF4 SNP g.18174 A > G genotypes. Expression of the NCF4 - TV transcript is highest in mammary samples from GG animals, followed by those from AG and AA individuals.
    Pspl3 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pspl3 vector/product/Thermo Fisher
    Average 79 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
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    80
    Addgene inc pspl3 expression vector
    In vivo and in vitro splicing analysis of the identified CDH23 alleles. ( A ) Analysis of the CDH23 mRNAs of whole blood samples (treated and untreated with cycloheximide, CHX) from patient and control by RT-PCR, showing the presence of aberrantly spliced CDH23 transcripts in addition to the wild-type transcript. Lane 1—patient sample (−CHX), Lane 2—patient sample (+CHX), Lane 3—control sample (−CHX), Lane 4—negative control. The band of 563 bp corresponded to the transcript with an in-frame skipping of exon 46. The band around 760–780 bp corresponded to the wild-type as well as to the aberrant transcripts with additional +7 and +13 nucleotides. Subsequent Sanger sequencing of cloned individual bands confirmed the insertion of 7 bp and 13 bp (774 pb, 780 pb bands) corresponding to each mutant allele. Transcripts bearing premature stop codon due to frameshift were only amplified in cycloheximide-treated blood samples (+CHX). ( B ) Schematic representation of the studied genomic region of CDH23 gene and the midigene construct for in vitro splicing assays (MGC1). The position of each mutation is indicated. The genomic region encompassing exons 45, 46, and 47 was cloned between the splice donor (SD) and acceptor (SA) sites within the <t>pSPL3</t> vector. ( C ) In vitro splicing assays in HEK293T cells transfected with either the wildtype (WT) or mutant CDH23 midigenes (MGC1-WT, MGC1-15A, and MGC1-9A, respectively) with or without cycloheximide treatment (+CHX). All constructs (MGC1-WT, MGC1-15A, and MGC1-9A) produced skipping of exon 46. CHX treatment in cells transfected with the mutant constructs increased the relative amplification of the aberrantly spliced transcripts. ( D ) Sanger sequence analysis of each transcript band confirmed the wild-type splicing event in cells transfected with MGC1-WT, in contrast to the addition of +13 and +7 nucleotides in exon 46 in cells transfected with MGC1-15A and MGC1-9A, respectively.
    Pspl3 Expression Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 80 stars, based on 5 article reviews
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    77
    Promega exon trapping vector pspl3
    In vivo and in vitro splicing analysis of the identified CDH23 alleles. ( A ) Analysis of the CDH23 mRNAs of whole blood samples (treated and untreated with cycloheximide, CHX) from patient and control by RT-PCR, showing the presence of aberrantly spliced CDH23 transcripts in addition to the wild-type transcript. Lane 1—patient sample (−CHX), Lane 2—patient sample (+CHX), Lane 3—control sample (−CHX), Lane 4—negative control. The band of 563 bp corresponded to the transcript with an in-frame skipping of exon 46. The band around 760–780 bp corresponded to the wild-type as well as to the aberrant transcripts with additional +7 and +13 nucleotides. Subsequent Sanger sequencing of cloned individual bands confirmed the insertion of 7 bp and 13 bp (774 pb, 780 pb bands) corresponding to each mutant allele. Transcripts bearing premature stop codon due to frameshift were only amplified in cycloheximide-treated blood samples (+CHX). ( B ) Schematic representation of the studied genomic region of CDH23 gene and the midigene construct for in vitro splicing assays (MGC1). The position of each mutation is indicated. The genomic region encompassing exons 45, 46, and 47 was cloned between the splice donor (SD) and acceptor (SA) sites within the <t>pSPL3</t> vector. ( C ) In vitro splicing assays in HEK293T cells transfected with either the wildtype (WT) or mutant CDH23 midigenes (MGC1-WT, MGC1-15A, and MGC1-9A, respectively) with or without cycloheximide treatment (+CHX). All constructs (MGC1-WT, MGC1-15A, and MGC1-9A) produced skipping of exon 46. CHX treatment in cells transfected with the mutant constructs increased the relative amplification of the aberrantly spliced transcripts. ( D ) Sanger sequence analysis of each transcript band confirmed the wild-type splicing event in cells transfected with MGC1-WT, in contrast to the addition of +13 and +7 nucleotides in exon 46 in cells transfected with MGC1-15A and MGC1-9A, respectively.
    Exon Trapping Vector Pspl3, supplied by Promega, used in various techniques. Bioz Stars score: 77/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 77 stars, based on 9 article reviews
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    93
    Thermo Fisher pspl3
    Constructs of minigenes and splicing analyses. ( A ) Partial genomic structure of CHRNA1 and two minigene constructs. pRBG4 minigene carries four native exons of CHRNA1 , whereas <t>pSPL3</t> minigene harbors only CHRNA1 exon P3A flanked by proprietary 5′
    Pspl3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pspl3/product/Thermo Fisher
    Average 93 stars, based on 116 article reviews
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    78
    Thermo Fisher splicing vector pspl3
    Constructs of minigenes and splicing analyses. ( A ) Partial genomic structure of CHRNA1 and two minigene constructs. pRBG4 minigene carries four native exons of CHRNA1 , whereas <t>pSPL3</t> minigene harbors only CHRNA1 exon P3A flanked by proprietary 5′
    Splicing Vector Pspl3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/splicing vector pspl3/product/Thermo Fisher
    Average 78 stars, based on 7 article reviews
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    splicing vector pspl3 - by Bioz Stars, 2020-02
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    78
    Thermo Fisher exon trap vector pspl3
    Constructs of minigenes and splicing analyses. ( A ) Partial genomic structure of CHRNA1 and two minigene constructs. pRBG4 minigene carries four native exons of CHRNA1 , whereas <t>pSPL3</t> minigene harbors only CHRNA1 exon P3A flanked by proprietary 5′
    Exon Trap Vector Pspl3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exon trap vector pspl3/product/Thermo Fisher
    Average 78 stars, based on 10 article reviews
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    exon trap vector pspl3 - by Bioz Stars, 2020-02
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    83
    Thermo Fisher double digested pspl3 plasmid vector
    Constructs of minigenes and splicing analyses. ( A ) Partial genomic structure of CHRNA1 and two minigene constructs. pRBG4 minigene carries four native exons of CHRNA1 , whereas <t>pSPL3</t> minigene harbors only CHRNA1 exon P3A flanked by proprietary 5′
    Double Digested Pspl3 Plasmid Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher exon trapping expression vector pspl3
    Representation of the <t>pSPL3-NF1-7</t> minigene construct. Genomic sequence from exon 7 of the NF1 gene along with 258 bp of 5' and 248 bp of 3' intronic flanking sequence was ligated into pSPL3 plasmid. Shaded sequences describe the nucleotide changes introduced independently by in vitro mutagenesis to obtain the mutated constructs.
    Exon Trapping Expression Vector Pspl3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exon trapping expression vector pspl3/product/Thermo Fisher
    Average 78 stars, based on 9 article reviews
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    78
    Thermo Fisher pspl3 based exon trapping system
    Splicing of Sgol2 and Tubd1 is responsive to changes in Tra2b concentration. ( A ) The murine and human versions of the genomic regions comprising the identified exons of Sgol2 and Tubd1 were cloned into the <t>pSPL3</t> exon trapping vector. ( B ) HEK293T cells were co-transfected with the pSPL3 minigene vector and siRNA specific for Tra2b or a TRA2B-GFP expression vector. Western Blot analysis shows efficiently reduced Tra2b protein levels and solid overexpression of TRA2B-GFP. ( C ) RNA was analyzed for exon inclusion after 48 h by semi-quantitative RT-PCR. ( D–G ) RT-PCR results were densitometrically quantified. Exon 4 of murine but not human Sgol2 is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 43% to 89%. Knock-down of Tra2b is insufficient to reduce Sgol2 exon4 splicing inclusion. Exon 4 of human but not murine Tubd1 is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 79% to 94%. Knock-down of Tra2b decreased inclusion of exon 4 from 79% to 71%. Nt, non-treated; scr, scrambled siRNA; si, siRNA against Tra2b ; n.s., non significant; error bars show the s.e.m.; significance levels are *p
    Pspl3 Based Exon Trapping System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Roche plasmid dna
    Splicing of Sgol2 and Tubd1 is responsive to changes in Tra2b concentration. ( A ) The murine and human versions of the genomic regions comprising the identified exons of Sgol2 and Tubd1 were cloned into the <t>pSPL3</t> exon trapping vector. ( B ) HEK293T cells were co-transfected with the pSPL3 minigene vector and siRNA specific for Tra2b or a TRA2B-GFP expression vector. Western Blot analysis shows efficiently reduced Tra2b protein levels and solid overexpression of TRA2B-GFP. ( C ) RNA was analyzed for exon inclusion after 48 h by semi-quantitative RT-PCR. ( D–G ) RT-PCR results were densitometrically quantified. Exon 4 of murine but not human Sgol2 is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 43% to 89%. Knock-down of Tra2b is insufficient to reduce Sgol2 exon4 splicing inclusion. Exon 4 of human but not murine Tubd1 is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 79% to 94%. Knock-down of Tra2b decreased inclusion of exon 4 from 79% to 71%. Nt, non-treated; scr, scrambled siRNA; si, siRNA against Tra2b ; n.s., non significant; error bars show the s.e.m.; significance levels are *p
    Plasmid Dna, supplied by Roche, used in various techniques. Bioz Stars score: 96/100, based on 7269 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 7269 article reviews
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    Image Search Results


    SNP g.18174 A > G induces aberrant NCF4 - TV splicing. (A) Schematic representation of the NCF4 mini-genes used in the functional splicing assay. The wild-type and mutant fragments contained 125 bp of intron 9 and 53 bp of exon 10; fragments harboring the A or G allele were separately cloned into the Eco RI and Xho I cloning sites of the pSPL3 vector. Two mini-gene expression vectors were transiently transfected into 293T cells. (B) RT-PCR analysis of the NCF4 spliced transcripts on a 2% agarose gel. RT-PCR products were amplified from the total RNA of 293T cells transfected with the wild-type and mutant (g.18174 A > G) NCF4 mini-gene constructs. The size of the RT-PCR product (441 bp) corresponded to the amplified portion of intron 9 (77 bp), the retained portion of intron 9 (48 bp), the amplification of exon 10 (53 bp), and the pSPL3 control plasmid (263 bp). The size of the RT-PCR product (393 bp) corresponded to the amplified portion of intron 9 (77 bp), the amplification of exon 10 (53 bp), and the pSPL3 control plasmid (263 bp). (C) Electrophoresis of RT-PCR products showing the presence and abundance of NCF4 - TV transcript in bovine mammary samples with three NCF4 SNP g.18174 A > G genotypes. Expression of the NCF4 - TV transcript is highest in mammary samples from GG animals, followed by those from AG and AA individuals.

    Journal: PLoS ONE

    Article Title: Role of an SNP in Alternative Splicing of Bovine NCF4 and Mastitis Susceptibility

    doi: 10.1371/journal.pone.0143705

    Figure Lengend Snippet: SNP g.18174 A > G induces aberrant NCF4 - TV splicing. (A) Schematic representation of the NCF4 mini-genes used in the functional splicing assay. The wild-type and mutant fragments contained 125 bp of intron 9 and 53 bp of exon 10; fragments harboring the A or G allele were separately cloned into the Eco RI and Xho I cloning sites of the pSPL3 vector. Two mini-gene expression vectors were transiently transfected into 293T cells. (B) RT-PCR analysis of the NCF4 spliced transcripts on a 2% agarose gel. RT-PCR products were amplified from the total RNA of 293T cells transfected with the wild-type and mutant (g.18174 A > G) NCF4 mini-gene constructs. The size of the RT-PCR product (441 bp) corresponded to the amplified portion of intron 9 (77 bp), the retained portion of intron 9 (48 bp), the amplification of exon 10 (53 bp), and the pSPL3 control plasmid (263 bp). The size of the RT-PCR product (393 bp) corresponded to the amplified portion of intron 9 (77 bp), the amplification of exon 10 (53 bp), and the pSPL3 control plasmid (263 bp). (C) Electrophoresis of RT-PCR products showing the presence and abundance of NCF4 - TV transcript in bovine mammary samples with three NCF4 SNP g.18174 A > G genotypes. Expression of the NCF4 - TV transcript is highest in mammary samples from GG animals, followed by those from AG and AA individuals.

    Article Snippet: After digestion with Eco RI and Xho I, the segment with the wild-type AA or the mutant type GG of the NCF4 gene were cloned into the pSPL3 vector (Invitrogen, CA, USA).

    Techniques: Functional Assay, Splicing Assay, Mutagenesis, Clone Assay, Plasmid Preparation, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Construct, Electrophoresis

    Splicing assays for the wild-type (WT) and mutant minigenes corresponding to the IDS nucleotide changes c.257C > T and c.241C > T in exon 3, and c.1122C > T in exon 8. (A, C) Diagrams of the reporter minigenes used in the functional splicing experiments. Normal and mutated genomic IDS sequences were cloned into the pcDNA3.1-myc or pSPL3 vectors to generate the indicated minigenes. Exons are shown by boxes and introns by straight lines. For all exonic alterations, the WT and mutant regions are shown and the specific changes marked by an arrow. (B, D) Wild-type and mutant minigenes were transfected into COS-7 and Hep3B cells and the splicing pattern analyzed by RT-PCR using the indicated vector-specific primers (arrows in diagrams A and C). Minigene expression of the splicing mutations in exon 3 (c.257C > T and c.241C > T) revealed two transcripts, a predominant one with exon 2 and a mutated exon 3, and a minor transcript of smaller size in which the first 44 nucleotides of exon 3 were missing. The WT minigene produced a single transcript of normal size (B). For the synonymous c.1122C > T change in exon 8, the mutant minigene showed a single transcript lacking the last 60bp of exon 8. The WT construct produced two bands, one corresponding to the transcript with exon 8 inserted, the other to a transcript resulting from an expected splicing event between the vector splice sites (D). A diagram of the bands characterized by sequence analysis is also provided. NC – negative control; V – vector sequence.

    Journal: Data in Brief

    Article Title: Data in support of a functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II

    doi: 10.1016/j.dib.2015.10.011

    Figure Lengend Snippet: Splicing assays for the wild-type (WT) and mutant minigenes corresponding to the IDS nucleotide changes c.257C > T and c.241C > T in exon 3, and c.1122C > T in exon 8. (A, C) Diagrams of the reporter minigenes used in the functional splicing experiments. Normal and mutated genomic IDS sequences were cloned into the pcDNA3.1-myc or pSPL3 vectors to generate the indicated minigenes. Exons are shown by boxes and introns by straight lines. For all exonic alterations, the WT and mutant regions are shown and the specific changes marked by an arrow. (B, D) Wild-type and mutant minigenes were transfected into COS-7 and Hep3B cells and the splicing pattern analyzed by RT-PCR using the indicated vector-specific primers (arrows in diagrams A and C). Minigene expression of the splicing mutations in exon 3 (c.257C > T and c.241C > T) revealed two transcripts, a predominant one with exon 2 and a mutated exon 3, and a minor transcript of smaller size in which the first 44 nucleotides of exon 3 were missing. The WT minigene produced a single transcript of normal size (B). For the synonymous c.1122C > T change in exon 8, the mutant minigene showed a single transcript lacking the last 60bp of exon 8. The WT construct produced two bands, one corresponding to the transcript with exon 8 inserted, the other to a transcript resulting from an expected splicing event between the vector splice sites (D). A diagram of the bands characterized by sequence analysis is also provided. NC – negative control; V – vector sequence.

    Article Snippet: Also, to functionally investigate the splicing defects caused by c.1122C > T in exon 8, wild-type (WT) and mutant minigenes were constructed in vector pSPL3 (Exon Trapping System, Life Technologies, Gibco, NY, USA) ( and C).

    Techniques: Mutagenesis, Functional Assay, Clone Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Expressing, Produced, Construct, Sequencing, Negative Control

    Mini-gene analysis of c.1679+1634A > G mutation. a) Schematic of pMG-in12-WT plasmid comprising a contiguous ~1.5kb region of CFTR containing part of intron 12, exon 13 and part of intron 13 cloned between the 5’ and 3’ exons of the pSPL3 vector. Exons are shown as boxes, introns as lines, and predicted transcript as thicker line shown underneath. Arrows below transcript represent pspRTfw and pspRTrv primers for RT-PCR. b) Electropherogram analysis of RT-PCR products (filled peaks) generated from HEK293T cells transfected with pMG-in12-WT and size markers (unfilled peaks). c) Schematic of pMG-in12-A > G mutation which differs from pMG-in12-WT by the A > G change shown with dotted arrow. The A > G change creates a pseudo exon (Ψ-EX) which contains an in-frame stop codon (TAA). The triangles flanking the Ψ-EX indicate target sites of Cas9/gRNAs encoded by pTandem-in12 vector. Arrows above DNA represent pspSEQfw and in12Drv primers for PCR. d) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in12-A > G. e) Schematic of pMG-in12-A > G mutation following targeted excision and repair. f) Agarose gel electrophoresis analysis of targeted deletions in pMG-in12-A > G measured by PCR. “-” lane is PCR products generated from HEK293T cells transfected with pMG-in12-A > G only, “+” lane is PCR products generated from HEK293T cells transfected with pMG-in12-A > G and pTandem-in12. The top band is generated from untargeted DNA, the bottom band is generated from DNA containing the targeted deletion, and the middle band is most likely heteroduplexes formed during the late stages of PCR; similar bands have been reported during PCR analysis of the CF-causing c.1521_1523delCTT deletion and verified as heteroduplexes [ 32 ]. g) Electropherogram analysis of RT-PCR products generated from HEK293T cells co-transfected with pMG-in12-A > G and pTandem-in12 vector.

    Journal: PLoS ONE

    Article Title: Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA

    doi: 10.1371/journal.pone.0184009

    Figure Lengend Snippet: Mini-gene analysis of c.1679+1634A > G mutation. a) Schematic of pMG-in12-WT plasmid comprising a contiguous ~1.5kb region of CFTR containing part of intron 12, exon 13 and part of intron 13 cloned between the 5’ and 3’ exons of the pSPL3 vector. Exons are shown as boxes, introns as lines, and predicted transcript as thicker line shown underneath. Arrows below transcript represent pspRTfw and pspRTrv primers for RT-PCR. b) Electropherogram analysis of RT-PCR products (filled peaks) generated from HEK293T cells transfected with pMG-in12-WT and size markers (unfilled peaks). c) Schematic of pMG-in12-A > G mutation which differs from pMG-in12-WT by the A > G change shown with dotted arrow. The A > G change creates a pseudo exon (Ψ-EX) which contains an in-frame stop codon (TAA). The triangles flanking the Ψ-EX indicate target sites of Cas9/gRNAs encoded by pTandem-in12 vector. Arrows above DNA represent pspSEQfw and in12Drv primers for PCR. d) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in12-A > G. e) Schematic of pMG-in12-A > G mutation following targeted excision and repair. f) Agarose gel electrophoresis analysis of targeted deletions in pMG-in12-A > G measured by PCR. “-” lane is PCR products generated from HEK293T cells transfected with pMG-in12-A > G only, “+” lane is PCR products generated from HEK293T cells transfected with pMG-in12-A > G and pTandem-in12. The top band is generated from untargeted DNA, the bottom band is generated from DNA containing the targeted deletion, and the middle band is most likely heteroduplexes formed during the late stages of PCR; similar bands have been reported during PCR analysis of the CF-causing c.1521_1523delCTT deletion and verified as heteroduplexes [ 32 ]. g) Electropherogram analysis of RT-PCR products generated from HEK293T cells co-transfected with pMG-in12-A > G and pTandem-in12 vector.

    Article Snippet: Target amplicons were sub-cloned in Xho I/Nhe I sites in a modified version (3053G > A to destroy a cryptic splice site) of the exon-trapping vector pSPL3 (Invitrogen, Dun Laoghaire, Ireland).

    Techniques: Mutagenesis, Plasmid Preparation, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Generated, Transfection, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Mini-gene analysis of c.3718-2477C > T mutation. a) Schematic of pMG-in22-WT plasmid comprising a contiguous ~3.2 kb region of CFTR containing part of intron 22, exon 23, and part of intron 23 cloned between the 5’ and 3’ exons of the pSPL3 vector. b) Electropherogram analysis of RT-PCR products (filled peaks) generated from HEK293T cells transfected with pMG-in22-WT. c) Schematic of pMG-in22-C > T mutation which differs from pMG-in22-WT by the C > T change shown with dotted arrow. The C > T change creates a pseudo exon (Ψ-EX) which contains an in-frame stop codon (TAA). The triangles flanking the Ψ-EX indicate target sites of Cas9/gRNAs encoded by pTandem-in22 vector. Arrows above DNA represent pspSEQfw and in22Drv primers for PCR. d) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in22-C > T. e) Schematic of pMG-in22-C > T mutation following targeted excision and repair. f) Agarose gel electrophoresis analysis of targeted deletions in pMG-in22-C > T measured by PCR. “-” lane is PCR products generated from HEK293T cells transfected with pMG-in22-C > T only, “+” lane is PCR products generated from HEK293T cells transfected with pMG-in22-C > T and pTandem-in22. g) Electropherogram analysis of RT-PCR products generated from HEK293T cells co-transfected with pMG-in22-C > T and pTandem-in22 vector.

    Journal: PLoS ONE

    Article Title: Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA

    doi: 10.1371/journal.pone.0184009

    Figure Lengend Snippet: Mini-gene analysis of c.3718-2477C > T mutation. a) Schematic of pMG-in22-WT plasmid comprising a contiguous ~3.2 kb region of CFTR containing part of intron 22, exon 23, and part of intron 23 cloned between the 5’ and 3’ exons of the pSPL3 vector. b) Electropherogram analysis of RT-PCR products (filled peaks) generated from HEK293T cells transfected with pMG-in22-WT. c) Schematic of pMG-in22-C > T mutation which differs from pMG-in22-WT by the C > T change shown with dotted arrow. The C > T change creates a pseudo exon (Ψ-EX) which contains an in-frame stop codon (TAA). The triangles flanking the Ψ-EX indicate target sites of Cas9/gRNAs encoded by pTandem-in22 vector. Arrows above DNA represent pspSEQfw and in22Drv primers for PCR. d) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in22-C > T. e) Schematic of pMG-in22-C > T mutation following targeted excision and repair. f) Agarose gel electrophoresis analysis of targeted deletions in pMG-in22-C > T measured by PCR. “-” lane is PCR products generated from HEK293T cells transfected with pMG-in22-C > T only, “+” lane is PCR products generated from HEK293T cells transfected with pMG-in22-C > T and pTandem-in22. g) Electropherogram analysis of RT-PCR products generated from HEK293T cells co-transfected with pMG-in22-C > T and pTandem-in22 vector.

    Article Snippet: Target amplicons were sub-cloned in Xho I/Nhe I sites in a modified version (3053G > A to destroy a cryptic splice site) of the exon-trapping vector pSPL3 (Invitrogen, Dun Laoghaire, Ireland).

    Techniques: Mutagenesis, Plasmid Preparation, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Generated, Transfection, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Mini-gene analysis of c.3140-26A > G mutation. a) Schematic of pMG-in19-WT plasmid comprising a contiguous ~2.0 kb region of CFTR containing part of intron 18, exon 19, all of intron 19, exon 20 and part of intron 20 cloned between the 5’ and 3’ exons of the pSPL3 vector. b) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in19-WT. c) Schematic of pMG-in19-A > G mutation which differs from pMG-in19-WT by the A > G change shown with dotted arrow. The A > G change extends exon 20 by 25 bp which shifts the reading frame and eventually results in-frame stop codon (TGA). The triangles indicate target sites of Cas9/gRNAs encoded by pTandem-in19 vector. Arrows above DNA represent in19Ufw and pspSEQrv primers for PCR. d) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in19-A > G. e) Schematic of pMG-in19-A > G mutation following targeted excision and repair. f) Agarose gel electrophoresis analysis of targeted deletions in pMG-in19-A > G measured by PCR. “-” lane is PCR products generated from HEK293T cells transfected with pMG-in19-A > G only, “+” lane is PCR products generated from HEK293T cells transfected with pMG-in19-A > G and pTandem-in12. g) Electropherogram analysis of RT-PCR products generated from HEK293T cells co-transfected with pMG-in19-A > G and pTandem-in19 vector.

    Journal: PLoS ONE

    Article Title: Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA

    doi: 10.1371/journal.pone.0184009

    Figure Lengend Snippet: Mini-gene analysis of c.3140-26A > G mutation. a) Schematic of pMG-in19-WT plasmid comprising a contiguous ~2.0 kb region of CFTR containing part of intron 18, exon 19, all of intron 19, exon 20 and part of intron 20 cloned between the 5’ and 3’ exons of the pSPL3 vector. b) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in19-WT. c) Schematic of pMG-in19-A > G mutation which differs from pMG-in19-WT by the A > G change shown with dotted arrow. The A > G change extends exon 20 by 25 bp which shifts the reading frame and eventually results in-frame stop codon (TGA). The triangles indicate target sites of Cas9/gRNAs encoded by pTandem-in19 vector. Arrows above DNA represent in19Ufw and pspSEQrv primers for PCR. d) Electropherogram analysis of RT-PCR products generated from HEK293T cells transfected with pMG-in19-A > G. e) Schematic of pMG-in19-A > G mutation following targeted excision and repair. f) Agarose gel electrophoresis analysis of targeted deletions in pMG-in19-A > G measured by PCR. “-” lane is PCR products generated from HEK293T cells transfected with pMG-in19-A > G only, “+” lane is PCR products generated from HEK293T cells transfected with pMG-in19-A > G and pTandem-in12. g) Electropherogram analysis of RT-PCR products generated from HEK293T cells co-transfected with pMG-in19-A > G and pTandem-in19 vector.

    Article Snippet: Target amplicons were sub-cloned in Xho I/Nhe I sites in a modified version (3053G > A to destroy a cryptic splice site) of the exon-trapping vector pSPL3 (Invitrogen, Dun Laoghaire, Ireland).

    Techniques: Mutagenesis, Plasmid Preparation, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Generated, Transfection, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Identification of F8 gene mutation and analysis of its effect on mRNA splicing. (a) Sanger sequencing of the hemophilia A patient. (b) Sanger sequencing of the patient's mother. (c) Sanger sequencing of a normal control. (d) cDNA sequencing of the patient's mother. (e) Agarose gel electrophoresis and sequencing diagram of minigene assay. The circle with a slash indicates a premature termination codon in exon 23. Arrows indicate mutations. M, markers; lanes one and 2, recombinant contains mutated genomic DNA fragment, mutant-pSPLS; lanes three and 4, recombinant contains normal genomic DNA sequence, wild-type-pSPL3; lanes five and 6, empty pSPL3 plasmid.

    Journal: Blood Coagulation & Fibrinolysis

    Article Title: An intronic mutation c.6430-3C > G in the F8 gene causes splicing efficiency and premature termination in hemophilia A

    doi: 10.1097/MBC.0000000000000730

    Figure Lengend Snippet: Identification of F8 gene mutation and analysis of its effect on mRNA splicing. (a) Sanger sequencing of the hemophilia A patient. (b) Sanger sequencing of the patient's mother. (c) Sanger sequencing of a normal control. (d) cDNA sequencing of the patient's mother. (e) Agarose gel electrophoresis and sequencing diagram of minigene assay. The circle with a slash indicates a premature termination codon in exon 23. Arrows indicate mutations. M, markers; lanes one and 2, recombinant contains mutated genomic DNA fragment, mutant-pSPLS; lanes three and 4, recombinant contains normal genomic DNA sequence, wild-type-pSPL3; lanes five and 6, empty pSPL3 plasmid.

    Article Snippet: We then constructed wild-type and mutant mini genes using pSPL3 plasmid (Invitrogen Corporation, Carlsbad, California, USA).

    Techniques: Mutagenesis, Sequencing, Agarose Gel Electrophoresis, Mini Gene Assay, Recombinant, Plasmid Preparation

    In vivo and in vitro splicing analysis of the identified CDH23 alleles. ( A ) Analysis of the CDH23 mRNAs of whole blood samples (treated and untreated with cycloheximide, CHX) from patient and control by RT-PCR, showing the presence of aberrantly spliced CDH23 transcripts in addition to the wild-type transcript. Lane 1—patient sample (−CHX), Lane 2—patient sample (+CHX), Lane 3—control sample (−CHX), Lane 4—negative control. The band of 563 bp corresponded to the transcript with an in-frame skipping of exon 46. The band around 760–780 bp corresponded to the wild-type as well as to the aberrant transcripts with additional +7 and +13 nucleotides. Subsequent Sanger sequencing of cloned individual bands confirmed the insertion of 7 bp and 13 bp (774 pb, 780 pb bands) corresponding to each mutant allele. Transcripts bearing premature stop codon due to frameshift were only amplified in cycloheximide-treated blood samples (+CHX). ( B ) Schematic representation of the studied genomic region of CDH23 gene and the midigene construct for in vitro splicing assays (MGC1). The position of each mutation is indicated. The genomic region encompassing exons 45, 46, and 47 was cloned between the splice donor (SD) and acceptor (SA) sites within the pSPL3 vector. ( C ) In vitro splicing assays in HEK293T cells transfected with either the wildtype (WT) or mutant CDH23 midigenes (MGC1-WT, MGC1-15A, and MGC1-9A, respectively) with or without cycloheximide treatment (+CHX). All constructs (MGC1-WT, MGC1-15A, and MGC1-9A) produced skipping of exon 46. CHX treatment in cells transfected with the mutant constructs increased the relative amplification of the aberrantly spliced transcripts. ( D ) Sanger sequence analysis of each transcript band confirmed the wild-type splicing event in cells transfected with MGC1-WT, in contrast to the addition of +13 and +7 nucleotides in exon 46 in cells transfected with MGC1-15A and MGC1-9A, respectively.

    Journal: Genes

    Article Title: Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1

    doi: 10.3390/genes10100732

    Figure Lengend Snippet: In vivo and in vitro splicing analysis of the identified CDH23 alleles. ( A ) Analysis of the CDH23 mRNAs of whole blood samples (treated and untreated with cycloheximide, CHX) from patient and control by RT-PCR, showing the presence of aberrantly spliced CDH23 transcripts in addition to the wild-type transcript. Lane 1—patient sample (−CHX), Lane 2—patient sample (+CHX), Lane 3—control sample (−CHX), Lane 4—negative control. The band of 563 bp corresponded to the transcript with an in-frame skipping of exon 46. The band around 760–780 bp corresponded to the wild-type as well as to the aberrant transcripts with additional +7 and +13 nucleotides. Subsequent Sanger sequencing of cloned individual bands confirmed the insertion of 7 bp and 13 bp (774 pb, 780 pb bands) corresponding to each mutant allele. Transcripts bearing premature stop codon due to frameshift were only amplified in cycloheximide-treated blood samples (+CHX). ( B ) Schematic representation of the studied genomic region of CDH23 gene and the midigene construct for in vitro splicing assays (MGC1). The position of each mutation is indicated. The genomic region encompassing exons 45, 46, and 47 was cloned between the splice donor (SD) and acceptor (SA) sites within the pSPL3 vector. ( C ) In vitro splicing assays in HEK293T cells transfected with either the wildtype (WT) or mutant CDH23 midigenes (MGC1-WT, MGC1-15A, and MGC1-9A, respectively) with or without cycloheximide treatment (+CHX). All constructs (MGC1-WT, MGC1-15A, and MGC1-9A) produced skipping of exon 46. CHX treatment in cells transfected with the mutant constructs increased the relative amplification of the aberrantly spliced transcripts. ( D ) Sanger sequence analysis of each transcript band confirmed the wild-type splicing event in cells transfected with MGC1-WT, in contrast to the addition of +13 and +7 nucleotides in exon 46 in cells transfected with MGC1-15A and MGC1-9A, respectively.

    Article Snippet: In Vitro Splicing Assays in HEK293T Cells For individual analysis of each of the CDH23 variants, (c.6050-15G > A and c.6050-9G > A), genomic fragments of 3751bp that spanned exons 45, 46, and 47 were subcloned into the HIV-tat intron of the pSPL3 expression vector (Addgene, Watertown, MA, USA).

    Techniques: In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction, Negative Control, Sequencing, Clone Assay, Mutagenesis, Amplification, Construct, Plasmid Preparation, Transfection, Produced

    Constructs of minigenes and splicing analyses. ( A ) Partial genomic structure of CHRNA1 and two minigene constructs. pRBG4 minigene carries four native exons of CHRNA1 , whereas pSPL3 minigene harbors only CHRNA1 exon P3A flanked by proprietary 5′

    Journal: Human Molecular Genetics

    Article Title: hnRNP H enhances skipping of a nonfunctional exon P3A in CHRNA1 and a mutation disrupting its binding causes congenital myasthenic syndrome

    doi: 10.1093/hmg/ddn305

    Figure Lengend Snippet: Constructs of minigenes and splicing analyses. ( A ) Partial genomic structure of CHRNA1 and two minigene constructs. pRBG4 minigene carries four native exons of CHRNA1 , whereas pSPL3 minigene harbors only CHRNA1 exon P3A flanked by proprietary 5′

    Article Snippet: We also constructed a minigene spanning exon P3A in the modified exon-trapping vector, pSPL3 (a discontinued product of Invitrogen), in which we introduced the CMV promoter in place of the SV40 promoter and also introduced Not I and Pac I restriction sites in the intron ( ).

    Techniques: Construct

    Representation of the pSPL3-NF1-7 minigene construct. Genomic sequence from exon 7 of the NF1 gene along with 258 bp of 5' and 248 bp of 3' intronic flanking sequence was ligated into pSPL3 plasmid. Shaded sequences describe the nucleotide changes introduced independently by in vitro mutagenesis to obtain the mutated constructs.

    Journal: BMC Medical Genetics

    Article Title: Functional analysis of splicing mutations in exon 7 of NF1 gene

    doi: 10.1186/1471-2350-8-4

    Figure Lengend Snippet: Representation of the pSPL3-NF1-7 minigene construct. Genomic sequence from exon 7 of the NF1 gene along with 258 bp of 5' and 248 bp of 3' intronic flanking sequence was ligated into pSPL3 plasmid. Shaded sequences describe the nucleotide changes introduced independently by in vitro mutagenesis to obtain the mutated constructs.

    Article Snippet: The exon trapping expression vector pSPL3 (Invitrogen Corporation, Carlsbad, CA) contains a replicon and Apr marker for growth in E. coli , an SV40 segment for replication and transcription in cos-1 cells, HIV-1 tat splicing signals, and a multiple cloning site.

    Techniques: Construct, Sequencing, Plasmid Preparation, In Vitro, Mutagenesis

    Splicing of Sgol2 and Tubd1 is responsive to changes in Tra2b concentration. ( A ) The murine and human versions of the genomic regions comprising the identified exons of Sgol2 and Tubd1 were cloned into the pSPL3 exon trapping vector. ( B ) HEK293T cells were co-transfected with the pSPL3 minigene vector and siRNA specific for Tra2b or a TRA2B-GFP expression vector. Western Blot analysis shows efficiently reduced Tra2b protein levels and solid overexpression of TRA2B-GFP. ( C ) RNA was analyzed for exon inclusion after 48 h by semi-quantitative RT-PCR. ( D–G ) RT-PCR results were densitometrically quantified. Exon 4 of murine but not human Sgol2 is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 43% to 89%. Knock-down of Tra2b is insufficient to reduce Sgol2 exon4 splicing inclusion. Exon 4 of human but not murine Tubd1 is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 79% to 94%. Knock-down of Tra2b decreased inclusion of exon 4 from 79% to 71%. Nt, non-treated; scr, scrambled siRNA; si, siRNA against Tra2b ; n.s., non significant; error bars show the s.e.m.; significance levels are *p

    Journal: PLoS ONE

    Article Title: Neuronal-Specific Deficiency of the Splicing Factor Tra2b Causes Apoptosis in Neurogenic Areas of the Developing Mouse Brain

    doi: 10.1371/journal.pone.0089020

    Figure Lengend Snippet: Splicing of Sgol2 and Tubd1 is responsive to changes in Tra2b concentration. ( A ) The murine and human versions of the genomic regions comprising the identified exons of Sgol2 and Tubd1 were cloned into the pSPL3 exon trapping vector. ( B ) HEK293T cells were co-transfected with the pSPL3 minigene vector and siRNA specific for Tra2b or a TRA2B-GFP expression vector. Western Blot analysis shows efficiently reduced Tra2b protein levels and solid overexpression of TRA2B-GFP. ( C ) RNA was analyzed for exon inclusion after 48 h by semi-quantitative RT-PCR. ( D–G ) RT-PCR results were densitometrically quantified. Exon 4 of murine but not human Sgol2 is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 43% to 89%. Knock-down of Tra2b is insufficient to reduce Sgol2 exon4 splicing inclusion. Exon 4 of human but not murine Tubd1 is responsive to increased concentrations of Tra2b as splicing inclusion significantly increased from 79% to 94%. Knock-down of Tra2b decreased inclusion of exon 4 from 79% to 71%. Nt, non-treated; scr, scrambled siRNA; si, siRNA against Tra2b ; n.s., non significant; error bars show the s.e.m.; significance levels are *p

    Article Snippet: Minigene Splicing Assay To assess splicing events we used the pSPL3-based exon trapping system from Life Technologies (Darmstadt, Germany).

    Techniques: Concentration Assay, Clone Assay, Plasmid Preparation, Transfection, Expressing, Western Blot, Over Expression, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction