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mtrr minigene  (GenScript corporation)

 
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    GenScript corporation mtrr minigene
    RHC-Glo splicing reporter <t>minigene</t> assay. Upper panel: schematic representation of the RHC-Glo splicing reporter minigene and the sub-cloned sequences. Lower panel: Agarose gel electrophoresis of RT-PCR minigene splicing products expressed in HEK293 cells. Inclusion or exclusion of exon 2 is indicated on the right. The gel pictures are representative results from two experiments.
    Mtrr Minigene, supplied by GenScript corporation, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/temporal+response+function+mtrf+toolbox/mtrr+minigene/pmc04482064-48-3-8
    Average 90 stars, based on 1 article reviews
    mtrr minigene - by Bioz Stars, 2026-11
    90/100 stars

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    1) Product Images from "Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells"

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv275

    RHC-Glo splicing reporter minigene assay. Upper panel: schematic representation of the RHC-Glo splicing reporter minigene and the sub-cloned sequences. Lower panel: Agarose gel electrophoresis of RT-PCR minigene splicing products expressed in HEK293 cells. Inclusion or exclusion of exon 2 is indicated on the right. The gel pictures are representative results from two experiments.
    Figure Legend Snippet: RHC-Glo splicing reporter minigene assay. Upper panel: schematic representation of the RHC-Glo splicing reporter minigene and the sub-cloned sequences. Lower panel: Agarose gel electrophoresis of RT-PCR minigene splicing products expressed in HEK293 cells. Inclusion or exclusion of exon 2 is indicated on the right. The gel pictures are representative results from two experiments.

    Techniques Used: Mini Gene Assay, Clone Assay, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    The balanced interplay between an hnRNP A1 binding ESS and two SRSF1 binding ESEs dictate MTRR pseudoexon activation. ( A ) Schematic representation of the sequences from MTRR -minigenes used and the nucleotide changes introduced. A pictogram of hnRNP A1 position weight matrix is shown above the putative ESS and the invariant position 2 of this motif is underscored. A pictogram of the SRSF1 position weight matrix is shown above the previously described ESE1 and the ACADM like ESE. Indicated positions 361, 362 and 365 are relative to the ACADM coding sequence. Nucleotide positions that were changed are underscored. ( B ) Splicing minigene assay. MTRR -minigenes were transiently transfected into HEK293. After RNA isolation the splicing products were analyzed by RT-PCR. The upper panel shows the average of pseudoexon inclusion from two measurements of each duplicate. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument. The lower panel shows a sample agarose gel electrophoresis displaying pseudoexon inclusion levels in the different cell lines. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inserted between minigene exons. Ψ marks the pseudoexon. ( C ) Binding of hnRNPA1 and SRSF1 proteins. Biotinylated RNA oligonucleotides were used in a pull-down experiment with HeLa nuclear extract followed by SDS PAGE and western blot analysis using antibodies against SRSF1 and hnRNP A1. Blank indicates a control lane from pull down without RNA oligonucleotides. NE is nuclear extract. The displayed blots are representative result from at least three pull-down experiments.
    Figure Legend Snippet: The balanced interplay between an hnRNP A1 binding ESS and two SRSF1 binding ESEs dictate MTRR pseudoexon activation. ( A ) Schematic representation of the sequences from MTRR -minigenes used and the nucleotide changes introduced. A pictogram of hnRNP A1 position weight matrix is shown above the putative ESS and the invariant position 2 of this motif is underscored. A pictogram of the SRSF1 position weight matrix is shown above the previously described ESE1 and the ACADM like ESE. Indicated positions 361, 362 and 365 are relative to the ACADM coding sequence. Nucleotide positions that were changed are underscored. ( B ) Splicing minigene assay. MTRR -minigenes were transiently transfected into HEK293. After RNA isolation the splicing products were analyzed by RT-PCR. The upper panel shows the average of pseudoexon inclusion from two measurements of each duplicate. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument. The lower panel shows a sample agarose gel electrophoresis displaying pseudoexon inclusion levels in the different cell lines. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inserted between minigene exons. Ψ marks the pseudoexon. ( C ) Binding of hnRNPA1 and SRSF1 proteins. Biotinylated RNA oligonucleotides were used in a pull-down experiment with HeLa nuclear extract followed by SDS PAGE and western blot analysis using antibodies against SRSF1 and hnRNP A1. Blank indicates a control lane from pull down without RNA oligonucleotides. NE is nuclear extract. The displayed blots are representative result from at least three pull-down experiments.

    Techniques Used: Binding Assay, Activation Assay, Sequencing, Mini Gene Assay, Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, SDS Page, Western Blot, Control

    SSO treatment in HEK293 cells transfected with wild-type, mutant and TCGGGA MTRR -minigenes. ( A ) Splicing minigene assay. MTRR -minigenes were transiently transfected into HEK293. 24 h after the cells were transfected with SSOs targeting either the 5′ss, 3′ss or both ESEs. After RNA isolation the splicing products were analyzed by RT-PCR. The upper panel shows a sample agarose gel electrophoresis displaying pseudoexon inclusion levels. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inclusion. Ψ marks the pseudoexon. ( B ) Quantification of pseudoexon inclusion. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument.
    Figure Legend Snippet: SSO treatment in HEK293 cells transfected with wild-type, mutant and TCGGGA MTRR -minigenes. ( A ) Splicing minigene assay. MTRR -minigenes were transiently transfected into HEK293. 24 h after the cells were transfected with SSOs targeting either the 5′ss, 3′ss or both ESEs. After RNA isolation the splicing products were analyzed by RT-PCR. The upper panel shows a sample agarose gel electrophoresis displaying pseudoexon inclusion levels. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inclusion. Ψ marks the pseudoexon. ( B ) Quantification of pseudoexon inclusion. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument.

    Techniques Used: Transfection, Mutagenesis, Mini Gene Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

    SSO treatment restores MTRR splicing in patient cells. ( A ) Fibroblasts from a patient harboring the c.903+469T>C mutation were transfected with SSOs that target either the 5′ss, the 3′ss, both ESEs (ESE-SSO), or a non-targeting sequence (NT). Control fibroblasts were treated in parallel. RNA was extracted after 48 h. A representative agarose gel electrophoresis of the RT-PCR products is shown. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inclusion between exon 6 and exon 7. Ψ marks the pseudoexon. ( B ) Quantification of pseudoexon inclusion in patient fibroblasts following SSO treatment. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument.
    Figure Legend Snippet: SSO treatment restores MTRR splicing in patient cells. ( A ) Fibroblasts from a patient harboring the c.903+469T>C mutation were transfected with SSOs that target either the 5′ss, the 3′ss, both ESEs (ESE-SSO), or a non-targeting sequence (NT). Control fibroblasts were treated in parallel. RNA was extracted after 48 h. A representative agarose gel electrophoresis of the RT-PCR products is shown. The lower bands represent correctly spliced exons, whereas the upper bands represent MTRR pseudoexon inclusion between exon 6 and exon 7. Ψ marks the pseudoexon. ( B ) Quantification of pseudoexon inclusion in patient fibroblasts following SSO treatment. Error bars represent the range. Quantification of PCR products was performed using a Fragment Analyzer instrument.

    Techniques Used: Mutagenesis, Transfection, Sequencing, Control, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    SSO treatment partially restores enzymatic activity of methionine synthase reductase (MTRR). Fibroblasts from a patient homozygous for the c.903+469T>C mutation were transfected with an SSO that target both ESEs (ESE-SSO) or a non-targeting sequence (Ctr). Fibroblasts were then labeled with [ 57 Co]cyanocabalamine and MTRR activity was determined indirectly by measuring methylcobalamine (MeCbl) synthesis (expressed as% of total labeled cobalamin derivatives). Adenosylcobalamine (AdoCbl) synthesis was measured as a control. The vertical lines represent the range of duplicate determinations in a representative experiment.
    Figure Legend Snippet: SSO treatment partially restores enzymatic activity of methionine synthase reductase (MTRR). Fibroblasts from a patient homozygous for the c.903+469T>C mutation were transfected with an SSO that target both ESEs (ESE-SSO) or a non-targeting sequence (Ctr). Fibroblasts were then labeled with [ 57 Co]cyanocabalamine and MTRR activity was determined indirectly by measuring methylcobalamine (MeCbl) synthesis (expressed as% of total labeled cobalamin derivatives). Adenosylcobalamine (AdoCbl) synthesis was measured as a control. The vertical lines represent the range of duplicate determinations in a representative experiment.

    Techniques Used: Activity Assay, Mutagenesis, Transfection, Sequencing, Labeling, Control

    Splicing model of the MTRR pseudoexon activation induced by the c.903+469T>C mutation. The wild-type sequence harbors a very weak SRSF1 binding ESE1, a flanking ACADM like SRSF1 binding ESE and a hnRNP A1 binding ESS. In the wild-type MTRR gene binding of hnRNP A1 to the TAGGGA high affinity ESS inhibits splicing because this initiates cooperative spreading of hnRNPA1 to weaker sites in a 3′-to-5′ direction thereby directly blocking access to the 5′ splice site and other splicing regulatory elements. Simultaneously binding of hnRNP A1 to the ESS inhibits binding of SRSF1 both to the very weak SRSF1 motif in ESE1 and to the ACADM -like ESE motif. This also affects recognition of the weak 5′ss by U1 snRNP, since this may be dependent on SRSF1 binding to the ESEs. When the c.903+469T>C mutation is present it changes the weak SRSF1 motif in ESE1 to a very strong site, which may act synergistically with the ACADM -like SRSF1 ESE to recruit U1snRNP to the weak 5′ss despite binding of hnRNP A1 to the flanking ESS does not seem to be affected. SRSF1 binding to the ESEs also antagonizes the cooperative spreading of hnRNPA1 binding from the TAGGGA high affinity ESS. Together this leads to pseudoexon activation and the abnormal splicing of the MTRR transcript observed in patients.
    Figure Legend Snippet: Splicing model of the MTRR pseudoexon activation induced by the c.903+469T>C mutation. The wild-type sequence harbors a very weak SRSF1 binding ESE1, a flanking ACADM like SRSF1 binding ESE and a hnRNP A1 binding ESS. In the wild-type MTRR gene binding of hnRNP A1 to the TAGGGA high affinity ESS inhibits splicing because this initiates cooperative spreading of hnRNPA1 to weaker sites in a 3′-to-5′ direction thereby directly blocking access to the 5′ splice site and other splicing regulatory elements. Simultaneously binding of hnRNP A1 to the ESS inhibits binding of SRSF1 both to the very weak SRSF1 motif in ESE1 and to the ACADM -like ESE motif. This also affects recognition of the weak 5′ss by U1 snRNP, since this may be dependent on SRSF1 binding to the ESEs. When the c.903+469T>C mutation is present it changes the weak SRSF1 motif in ESE1 to a very strong site, which may act synergistically with the ACADM -like SRSF1 ESE to recruit U1snRNP to the weak 5′ss despite binding of hnRNP A1 to the flanking ESS does not seem to be affected. SRSF1 binding to the ESEs also antagonizes the cooperative spreading of hnRNPA1 binding from the TAGGGA high affinity ESS. Together this leads to pseudoexon activation and the abnormal splicing of the MTRR transcript observed in patients.

    Techniques Used: Activation Assay, Mutagenesis, Sequencing, Binding Assay, Blocking Assay

    Related Articles

    Mini Gene Assay:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Clone Assay:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Agarose Gel Electrophoresis:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Binding Assay:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Activation Assay:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Sequencing:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Transfection:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Isolation:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    SDS Page:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Western Blot:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Control:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Mutagenesis:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Activity Assay:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Labeling:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to

    Blocking Assay:

    Article Title: Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells
    Article Snippet: MTRR minigene (beta-globin variant) has previously been described ( ). .. Mutations in the MTRR minigene were introduced by GeneScript Inc. (GenScript, Piscataway, NJ, USA). .. Wild-type and mutant double stranded DNA oligonucleotides corresponding to



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