pbr322 backbone  (New England Biolabs)


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    pBR322 DNA BstN I Digest
    Description:
    pBR322 DNA BstN I Digest 250 gel lanes
    Catalog Number:
    N3031L
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    302
    Category:
    DNA Molecular Weight Markers
    Size:
    250 gel lanes
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    New England Biolabs pbr322 backbone
    pBR322 DNA BstN I Digest
    pBR322 DNA BstN I Digest 250 gel lanes
    https://www.bioz.com/result/pbr322 backbone/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbr322 backbone - by Bioz Stars, 2021-06
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    1) Product Images from "Ablation of Arginylation in the Mouse N-End Rule Pathway: Loss of Fat, Higher Metabolic Rate, Damaged Spermatogenesis, and Neurological Perturbations"

    Article Title: Ablation of Arginylation in the Mouse N-End Rule Pathway: Loss of Fat, Higher Metabolic Rate, Damaged Spermatogenesis, and Neurological Perturbations

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007757

    Genomic configurations at the Ate1 locus of Cre-lox -based mouse strains constructed in the present work. (A) The 5′ end of the previously produced unconditional Ate1 − allele [10] , in which the Ate1 exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P Ate1 promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P PGK promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse Ate1 , indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of Ate1 homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of Ate1 exon 2, a “floxed”-hygromycin-resistance ( hph ) cassette, expressed from the P PGK promoter (blue arrow between two red triangles) downstream of Ate1 exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P HSV promoter (yellow arrow). Wavy line indicates an abutting sequence of the pBR322 plasmid DNA. (D) The tri-lox Ate1 allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active Ate1 flox allele), whereas “flox-off” indicates a configuration depicted in panel F (the null Ate1 − allele). The functionally active, “flox-on” ( Ate1 flox ) allele, obtained by the removal of the hph cassette, using the in vivo expression of Cre-recombinase driven by the P EIIA promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” ( Ate1 − ) allele obtained by the inducible expression of CreER recombinase from the P Cagg promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and Materials and Methods ). H, approximate locations of HindIII sites used in Southern analyses with DNA probe A (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe D (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes A and D , respectively. (G) Southern hybridization analysis using DNA probe A and HindIII-digested genomic DNA. The wt Ate1 allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed [10] unconditionally null Ate1 − allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on ( Ate1 flox ) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, Ate1 +/+ ; lane 2, Ate1 +/− ; lane 3, Ate1 +/− ; lane 4, Ate1 flox/− . (H) Southern hybridization analysis using DNA probe D (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed [10] unconditionally null Ate1 − allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type Ate1 allele and the flox-on ( Ate1 flox ) allele yield the 9.7 kB fragment, whereas the null flox-off ( Ate1 − ) allele yields the characteristic 3.8 kb fragment. The use of DNA probe D and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated Ate1 flox/− ; CaggCreER mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off ( Ate1 − ) allele. For example, whereas no flox-on ( Ate1 flox ) allele could be detected in the kidney and brain of Ate1 flox/− ; CaggCreER mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on ( Ate1 flox ) and flox-off ( Ate1 − ) alleles were present in the heart of TM-treated Ate1 flox/− ; CaggCreER mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed [10] Ate1 +/− mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated Ate1 flox/− ; CaggCreER mice. Lane 8, same as lane 7, but from a TM-treated Ate1 flox/− mouse (lacking the CaggCreER transgene).
    Figure Legend Snippet: Genomic configurations at the Ate1 locus of Cre-lox -based mouse strains constructed in the present work. (A) The 5′ end of the previously produced unconditional Ate1 − allele [10] , in which the Ate1 exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P Ate1 promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P PGK promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse Ate1 , indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of Ate1 homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of Ate1 exon 2, a “floxed”-hygromycin-resistance ( hph ) cassette, expressed from the P PGK promoter (blue arrow between two red triangles) downstream of Ate1 exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P HSV promoter (yellow arrow). Wavy line indicates an abutting sequence of the pBR322 plasmid DNA. (D) The tri-lox Ate1 allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active Ate1 flox allele), whereas “flox-off” indicates a configuration depicted in panel F (the null Ate1 − allele). The functionally active, “flox-on” ( Ate1 flox ) allele, obtained by the removal of the hph cassette, using the in vivo expression of Cre-recombinase driven by the P EIIA promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” ( Ate1 − ) allele obtained by the inducible expression of CreER recombinase from the P Cagg promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and Materials and Methods ). H, approximate locations of HindIII sites used in Southern analyses with DNA probe A (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe D (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes A and D , respectively. (G) Southern hybridization analysis using DNA probe A and HindIII-digested genomic DNA. The wt Ate1 allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed [10] unconditionally null Ate1 − allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on ( Ate1 flox ) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, Ate1 +/+ ; lane 2, Ate1 +/− ; lane 3, Ate1 +/− ; lane 4, Ate1 flox/− . (H) Southern hybridization analysis using DNA probe D (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed [10] unconditionally null Ate1 − allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type Ate1 allele and the flox-on ( Ate1 flox ) allele yield the 9.7 kB fragment, whereas the null flox-off ( Ate1 − ) allele yields the characteristic 3.8 kb fragment. The use of DNA probe D and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated Ate1 flox/− ; CaggCreER mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off ( Ate1 − ) allele. For example, whereas no flox-on ( Ate1 flox ) allele could be detected in the kidney and brain of Ate1 flox/− ; CaggCreER mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on ( Ate1 flox ) and flox-off ( Ate1 − ) alleles were present in the heart of TM-treated Ate1 flox/− ; CaggCreER mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed [10] Ate1 +/− mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated Ate1 flox/− ; CaggCreER mice. Lane 8, same as lane 7, but from a TM-treated Ate1 flox/− mouse (lacking the CaggCreER transgene).

    Techniques Used: Construct, Produced, Selection, Marker, Sequencing, Plasmid Preparation, In Vivo, Expressing, Hybridization, Mouse Assay

    Related Articles

    Purification:

    Article Title: The Fitness Landscapes of cis-Acting Binding Sites in Different Promoter and Environmental Contexts
    Article Snippet: Solexa sample prep and sequencing Conversion of pBR322 to pBR322 destroyed a Hind III site that overlapped the first two bases of the hexamer. .. Libraries that contained the wild type ( TTTAAT ) were digested with Hind III and Pvu I (NEB) for 2 hours at C. pBR322 libraries were digested with Cla I and Pvu I (NEB) for 2 hours at C. base pair fragments were gel purified for all four libraries using the QIAquick gel extraction kit. .. Excised fragments from all four promoter libraries, selected at a single tetracycline concentration, were mixed at equal concentration.

    Article Title: Peptides Containing Cyclin/Cdk-Nuclear Localization Signal Motifs Derived from Viral Initiator Proteins Bind to DNA When Unphosphorylated
    Article Snippet: T4 polynucleotide kinase was purchased from Gibco-BRL. .. Plasmid pBR322 DNA, used as competitor DNA, was purified according to standard procedures ( ) and digested with Hae III purchased from New England Biolabs. .. Oligonucleotides were synthesized on an Applied Biosystems 394 DNA synthesizer, purified by electrophoresis through 10% urea-polyacrylamide gels, and isolated as described previously ( , ).

    Gel Extraction:

    Article Title: The Fitness Landscapes of cis-Acting Binding Sites in Different Promoter and Environmental Contexts
    Article Snippet: Solexa sample prep and sequencing Conversion of pBR322 to pBR322 destroyed a Hind III site that overlapped the first two bases of the hexamer. .. Libraries that contained the wild type ( TTTAAT ) were digested with Hind III and Pvu I (NEB) for 2 hours at C. pBR322 libraries were digested with Cla I and Pvu I (NEB) for 2 hours at C. base pair fragments were gel purified for all four libraries using the QIAquick gel extraction kit. .. Excised fragments from all four promoter libraries, selected at a single tetracycline concentration, were mixed at equal concentration.

    Plasmid Preparation:

    Article Title: Peptides Containing Cyclin/Cdk-Nuclear Localization Signal Motifs Derived from Viral Initiator Proteins Bind to DNA When Unphosphorylated
    Article Snippet: T4 polynucleotide kinase was purchased from Gibco-BRL. .. Plasmid pBR322 DNA, used as competitor DNA, was purified according to standard procedures ( ) and digested with Hae III purchased from New England Biolabs. .. Oligonucleotides were synthesized on an Applied Biosystems 394 DNA synthesizer, purified by electrophoresis through 10% urea-polyacrylamide gels, and isolated as described previously ( , ).

    Article Title: Ablation of Arginylation in the Mouse N-End Rule Pathway: Loss of Fat, Higher Metabolic Rate, Damaged Spermatogenesis, and Neurological Perturbations
    Article Snippet: .. The entire ∼12 kb HindIII fragment and a part of the ∼2.9 kb fragment were modified as described below and assembled into a final ∼22.5 kb targeting vector consisting of the following parts ( ): ( i ) pBR322 backbone (New England Biolabs, Ipswich, MA); ( ii ) a ∼6.3 kb “long arm” of Ate1 homology containing the Ate1 exon 1a, the bidirectional PAte1 promoter , and exon 1b; ( iii ) A single loxP site ∼300 bp upstream of Ate1 exon 2; ( iv ) a ∼2 kb fragment that contains, 50 bp downstream of Ate1 exon 4, a “floxed” Hph (hygromycin) antibiotic-resistance marker, expressed from the PPGK promoter ; ( v ) a ∼1.2 kb “short arm” of Ate1 homology that spans most of the intron between exons 4 and 5; ( vi ) a gene encoding HSV-TK (herpes simplex virus thymidine kinase), expressed from the PPGK promoter. .. The targeting vector was linearized with BamHI and electroporated into CJ7 embryonic stem (ES) cells (a gift from Dr. Thomas Gridley, formerly of Jackson Laboratories, Bar Harbor, ME).

    Article Title: Sensitivity of human prostate cancer cells to chemotherapeutic drugs depends on EndoG expression regulated by promoter methylation
    Article Snippet: This assay was used for the measurement of endonuclease activity as previously described [ ]. .. The activity was measured in 20 μl samples containing 1 μg plasmid pBR322 DNA (New England Biolabs, Beverly, MA), 10 mM Tris-HCl, pH 7.7, 25 μg/ml bovine serum albumin V fraction, 0.5 mM dithiothreitol, 5 mM MnCl2, and 2 μl of cell extract in serial dilutions 1:5. .. After 1 h incubation at 37°C, the reaction was stopped by the addition of 5 μl 1% SDS and 100 mM EDTA.

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases
    Article Snippet: One unit was defined as the amount of endonuclease capable of converting 1 μg covalently closed supercoiled plasmid DNA to open circular, linear, or digested DNA in 1 h at 37°C. .. Endonuclease activity was measured the same way as above in samples containing serially diluted protein (1:5), 1 μg plasmid pBR322 DNA (New England Biolabs), 2 mM CaCl2 , 5 mM MgCl2 , 10 mM Tris-HCl, pH 7.4, and 0.5 mM dithiothreitol to determine the Ca/Mg-dependent (primarily DNase I) endonuclease activity or 5 mM MnCl2 , 10 mM Tris-HCl (pH 7.4), and 0.5 mM dithiothreitol for the Mn-dependent (mainly EndoG) activity. .. As opposed to PIA, zymogram gel electrophoresis, previously used by us to assess DNase I activity (Basnakian et al ., ), was not applicable for EndoG due to low specific activity of the enzyme in the used tubular epithelial cells.

    Modification:

    Article Title: Ablation of Arginylation in the Mouse N-End Rule Pathway: Loss of Fat, Higher Metabolic Rate, Damaged Spermatogenesis, and Neurological Perturbations
    Article Snippet: .. The entire ∼12 kb HindIII fragment and a part of the ∼2.9 kb fragment were modified as described below and assembled into a final ∼22.5 kb targeting vector consisting of the following parts ( ): ( i ) pBR322 backbone (New England Biolabs, Ipswich, MA); ( ii ) a ∼6.3 kb “long arm” of Ate1 homology containing the Ate1 exon 1a, the bidirectional PAte1 promoter , and exon 1b; ( iii ) A single loxP site ∼300 bp upstream of Ate1 exon 2; ( iv ) a ∼2 kb fragment that contains, 50 bp downstream of Ate1 exon 4, a “floxed” Hph (hygromycin) antibiotic-resistance marker, expressed from the PPGK promoter ; ( v ) a ∼1.2 kb “short arm” of Ate1 homology that spans most of the intron between exons 4 and 5; ( vi ) a gene encoding HSV-TK (herpes simplex virus thymidine kinase), expressed from the PPGK promoter. .. The targeting vector was linearized with BamHI and electroporated into CJ7 embryonic stem (ES) cells (a gift from Dr. Thomas Gridley, formerly of Jackson Laboratories, Bar Harbor, ME).

    Marker:

    Article Title: Ablation of Arginylation in the Mouse N-End Rule Pathway: Loss of Fat, Higher Metabolic Rate, Damaged Spermatogenesis, and Neurological Perturbations
    Article Snippet: .. The entire ∼12 kb HindIII fragment and a part of the ∼2.9 kb fragment were modified as described below and assembled into a final ∼22.5 kb targeting vector consisting of the following parts ( ): ( i ) pBR322 backbone (New England Biolabs, Ipswich, MA); ( ii ) a ∼6.3 kb “long arm” of Ate1 homology containing the Ate1 exon 1a, the bidirectional PAte1 promoter , and exon 1b; ( iii ) A single loxP site ∼300 bp upstream of Ate1 exon 2; ( iv ) a ∼2 kb fragment that contains, 50 bp downstream of Ate1 exon 4, a “floxed” Hph (hygromycin) antibiotic-resistance marker, expressed from the PPGK promoter ; ( v ) a ∼1.2 kb “short arm” of Ate1 homology that spans most of the intron between exons 4 and 5; ( vi ) a gene encoding HSV-TK (herpes simplex virus thymidine kinase), expressed from the PPGK promoter. .. The targeting vector was linearized with BamHI and electroporated into CJ7 embryonic stem (ES) cells (a gift from Dr. Thomas Gridley, formerly of Jackson Laboratories, Bar Harbor, ME).

    Article Title: In Vitro Susceptibility Testing of Borrelia burgdorferi Sensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy
    Article Snippet: PCR products (10 μl) were digested with 5 U of MseI (New England Biolabs) in a total volume of 20 μl. .. The resulting species-specific PCR-RFLP patterns were analyzed by electrophoresis on a 12% polyacrylamide gel stained with ethidium bromide, and the fragment size was determined by comparison to DNA fragments of a pBR322 molecular weight standard digested with MspI (New England Biolabs) and a 123-bp marker (Gibco-BRL Life Technologies). ..

    Activity Assay:

    Article Title: Sensitivity of human prostate cancer cells to chemotherapeutic drugs depends on EndoG expression regulated by promoter methylation
    Article Snippet: This assay was used for the measurement of endonuclease activity as previously described [ ]. .. The activity was measured in 20 μl samples containing 1 μg plasmid pBR322 DNA (New England Biolabs, Beverly, MA), 10 mM Tris-HCl, pH 7.7, 25 μg/ml bovine serum albumin V fraction, 0.5 mM dithiothreitol, 5 mM MnCl2, and 2 μl of cell extract in serial dilutions 1:5. .. After 1 h incubation at 37°C, the reaction was stopped by the addition of 5 μl 1% SDS and 100 mM EDTA.

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases
    Article Snippet: One unit was defined as the amount of endonuclease capable of converting 1 μg covalently closed supercoiled plasmid DNA to open circular, linear, or digested DNA in 1 h at 37°C. .. Endonuclease activity was measured the same way as above in samples containing serially diluted protein (1:5), 1 μg plasmid pBR322 DNA (New England Biolabs), 2 mM CaCl2 , 5 mM MgCl2 , 10 mM Tris-HCl, pH 7.4, and 0.5 mM dithiothreitol to determine the Ca/Mg-dependent (primarily DNase I) endonuclease activity or 5 mM MnCl2 , 10 mM Tris-HCl (pH 7.4), and 0.5 mM dithiothreitol for the Mn-dependent (mainly EndoG) activity. .. As opposed to PIA, zymogram gel electrophoresis, previously used by us to assess DNase I activity (Basnakian et al ., ), was not applicable for EndoG due to low specific activity of the enzyme in the used tubular epithelial cells.

    Polymerase Chain Reaction:

    Article Title: In Vitro Susceptibility Testing of Borrelia burgdorferi Sensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy
    Article Snippet: PCR products (10 μl) were digested with 5 U of MseI (New England Biolabs) in a total volume of 20 μl. .. The resulting species-specific PCR-RFLP patterns were analyzed by electrophoresis on a 12% polyacrylamide gel stained with ethidium bromide, and the fragment size was determined by comparison to DNA fragments of a pBR322 molecular weight standard digested with MspI (New England Biolabs) and a 123-bp marker (Gibco-BRL Life Technologies). ..

    Electrophoresis:

    Article Title: In Vitro Susceptibility Testing of Borrelia burgdorferi Sensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy
    Article Snippet: PCR products (10 μl) were digested with 5 U of MseI (New England Biolabs) in a total volume of 20 μl. .. The resulting species-specific PCR-RFLP patterns were analyzed by electrophoresis on a 12% polyacrylamide gel stained with ethidium bromide, and the fragment size was determined by comparison to DNA fragments of a pBR322 molecular weight standard digested with MspI (New England Biolabs) and a 123-bp marker (Gibco-BRL Life Technologies). ..

    Staining:

    Article Title: In Vitro Susceptibility Testing of Borrelia burgdorferi Sensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy
    Article Snippet: PCR products (10 μl) were digested with 5 U of MseI (New England Biolabs) in a total volume of 20 μl. .. The resulting species-specific PCR-RFLP patterns were analyzed by electrophoresis on a 12% polyacrylamide gel stained with ethidium bromide, and the fragment size was determined by comparison to DNA fragments of a pBR322 molecular weight standard digested with MspI (New England Biolabs) and a 123-bp marker (Gibco-BRL Life Technologies). ..

    Molecular Weight:

    Article Title: In Vitro Susceptibility Testing of Borrelia burgdorferi Sensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy
    Article Snippet: PCR products (10 μl) were digested with 5 U of MseI (New England Biolabs) in a total volume of 20 μl. .. The resulting species-specific PCR-RFLP patterns were analyzed by electrophoresis on a 12% polyacrylamide gel stained with ethidium bromide, and the fragment size was determined by comparison to DNA fragments of a pBR322 molecular weight standard digested with MspI (New England Biolabs) and a 123-bp marker (Gibco-BRL Life Technologies). ..

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    New England Biolabs pbr322 backbone
    Genomic configurations at the Ate1 locus of Cre-lox -based mouse strains constructed in the present work. (A) The 5′ end of the previously produced unconditional Ate1 − allele [10] , in which the Ate1 exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P Ate1 promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P PGK promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse Ate1 , indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of Ate1 homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of Ate1 exon 2, a “floxed”-hygromycin-resistance ( hph ) cassette, expressed from the P PGK promoter (blue arrow between two red triangles) downstream of Ate1 exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P HSV promoter (yellow arrow). Wavy line indicates an abutting sequence of the <t>pBR322</t> plasmid DNA. (D) The tri-lox Ate1 allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active Ate1 flox allele), whereas “flox-off” indicates a configuration depicted in panel F (the null Ate1 − allele). The functionally active, “flox-on” ( Ate1 flox ) allele, obtained by the removal of the hph cassette, using the in vivo expression of Cre-recombinase driven by the P EIIA promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” ( Ate1 − ) allele obtained by the inducible expression of CreER recombinase from the P Cagg promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and Materials and Methods ). H, approximate locations of HindIII sites used in Southern analyses with DNA probe A (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe D (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes A and D , respectively. (G) Southern hybridization analysis using DNA probe A and HindIII-digested genomic DNA. The wt Ate1 allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed [10] unconditionally null Ate1 − allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on ( Ate1 flox ) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, Ate1 +/+ ; lane 2, Ate1 +/− ; lane 3, Ate1 +/− ; lane 4, Ate1 flox/− . (H) Southern hybridization analysis using DNA probe D (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed [10] unconditionally null Ate1 − allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type Ate1 allele and the flox-on ( Ate1 flox ) allele yield the 9.7 kB fragment, whereas the null flox-off ( Ate1 − ) allele yields the characteristic 3.8 kb fragment. The use of DNA probe D and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated Ate1 flox/− ; CaggCreER mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off ( Ate1 − ) allele. For example, whereas no flox-on ( Ate1 flox ) allele could be detected in the kidney and brain of Ate1 flox/− ; CaggCreER mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on ( Ate1 flox ) and flox-off ( Ate1 − ) alleles were present in the heart of TM-treated Ate1 flox/− ; CaggCreER mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed [10] Ate1 +/− mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated Ate1 flox/− ; CaggCreER mice. Lane 8, same as lane 7, but from a TM-treated Ate1 flox/− mouse (lacking the CaggCreER transgene).
    Pbr322 Backbone, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    New England Biolabs pbr322 plasmid backbone
    Genomic configurations at the Ate1 locus of Cre-lox -based mouse strains constructed in the present work. (A) The 5′ end of the previously produced unconditional Ate1 − allele [10] , in which the Ate1 exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P Ate1 promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P PGK promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse Ate1 , indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of Ate1 homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of Ate1 exon 2, a “floxed”-hygromycin-resistance ( hph ) cassette, expressed from the P PGK promoter (blue arrow between two red triangles) downstream of Ate1 exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P HSV promoter (yellow arrow). Wavy line indicates an abutting sequence of the <t>pBR322</t> plasmid DNA. (D) The tri-lox Ate1 allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active Ate1 flox allele), whereas “flox-off” indicates a configuration depicted in panel F (the null Ate1 − allele). The functionally active, “flox-on” ( Ate1 flox ) allele, obtained by the removal of the hph cassette, using the in vivo expression of Cre-recombinase driven by the P EIIA promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” ( Ate1 − ) allele obtained by the inducible expression of CreER recombinase from the P Cagg promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and Materials and Methods ). H, approximate locations of HindIII sites used in Southern analyses with DNA probe A (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe D (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes A and D , respectively. (G) Southern hybridization analysis using DNA probe A and HindIII-digested genomic DNA. The wt Ate1 allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed [10] unconditionally null Ate1 − allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on ( Ate1 flox ) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, Ate1 +/+ ; lane 2, Ate1 +/− ; lane 3, Ate1 +/− ; lane 4, Ate1 flox/− . (H) Southern hybridization analysis using DNA probe D (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed [10] unconditionally null Ate1 − allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type Ate1 allele and the flox-on ( Ate1 flox ) allele yield the 9.7 kB fragment, whereas the null flox-off ( Ate1 − ) allele yields the characteristic 3.8 kb fragment. The use of DNA probe D and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated Ate1 flox/− ; CaggCreER mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off ( Ate1 − ) allele. For example, whereas no flox-on ( Ate1 flox ) allele could be detected in the kidney and brain of Ate1 flox/− ; CaggCreER mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on ( Ate1 flox ) and flox-off ( Ate1 − ) alleles were present in the heart of TM-treated Ate1 flox/− ; CaggCreER mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed [10] Ate1 +/− mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated Ate1 flox/− ; CaggCreER mice. Lane 8, same as lane 7, but from a TM-treated Ate1 flox/− mouse (lacking the CaggCreER transgene).
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    98
    New England Biolabs pbr322 vector backbone
    Genomic configurations at the Ate1 locus of Cre-lox -based mouse strains constructed in the present work. (A) The 5′ end of the previously produced unconditional Ate1 − allele [10] , in which the Ate1 exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P Ate1 promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P PGK promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse Ate1 , indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of Ate1 homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of Ate1 exon 2, a “floxed”-hygromycin-resistance ( hph ) cassette, expressed from the P PGK promoter (blue arrow between two red triangles) downstream of Ate1 exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P HSV promoter (yellow arrow). Wavy line indicates an abutting sequence of the <t>pBR322</t> plasmid DNA. (D) The tri-lox Ate1 allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active Ate1 flox allele), whereas “flox-off” indicates a configuration depicted in panel F (the null Ate1 − allele). The functionally active, “flox-on” ( Ate1 flox ) allele, obtained by the removal of the hph cassette, using the in vivo expression of Cre-recombinase driven by the P EIIA promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” ( Ate1 − ) allele obtained by the inducible expression of CreER recombinase from the P Cagg promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and Materials and Methods ). H, approximate locations of HindIII sites used in Southern analyses with DNA probe A (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe D (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes A and D , respectively. (G) Southern hybridization analysis using DNA probe A and HindIII-digested genomic DNA. The wt Ate1 allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed [10] unconditionally null Ate1 − allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on ( Ate1 flox ) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, Ate1 +/+ ; lane 2, Ate1 +/− ; lane 3, Ate1 +/− ; lane 4, Ate1 flox/− . (H) Southern hybridization analysis using DNA probe D (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed [10] unconditionally null Ate1 − allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type Ate1 allele and the flox-on ( Ate1 flox ) allele yield the 9.7 kB fragment, whereas the null flox-off ( Ate1 − ) allele yields the characteristic 3.8 kb fragment. The use of DNA probe D and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated Ate1 flox/− ; CaggCreER mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off ( Ate1 − ) allele. For example, whereas no flox-on ( Ate1 flox ) allele could be detected in the kidney and brain of Ate1 flox/− ; CaggCreER mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on ( Ate1 flox ) and flox-off ( Ate1 − ) alleles were present in the heart of TM-treated Ate1 flox/− ; CaggCreER mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed [10] Ate1 +/− mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated Ate1 flox/− ; CaggCreER mice. Lane 8, same as lane 7, but from a TM-treated Ate1 flox/− mouse (lacking the CaggCreER transgene).
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    Genomic configurations at the Ate1 locus of Cre-lox -based mouse strains constructed in the present work. (A) The 5′ end of the previously produced unconditional Ate1 − allele [10] , in which the Ate1 exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P Ate1 promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P PGK promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse Ate1 , indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of Ate1 homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of Ate1 exon 2, a “floxed”-hygromycin-resistance ( hph ) cassette, expressed from the P PGK promoter (blue arrow between two red triangles) downstream of Ate1 exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P HSV promoter (yellow arrow). Wavy line indicates an abutting sequence of the pBR322 plasmid DNA. (D) The tri-lox Ate1 allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active Ate1 flox allele), whereas “flox-off” indicates a configuration depicted in panel F (the null Ate1 − allele). The functionally active, “flox-on” ( Ate1 flox ) allele, obtained by the removal of the hph cassette, using the in vivo expression of Cre-recombinase driven by the P EIIA promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” ( Ate1 − ) allele obtained by the inducible expression of CreER recombinase from the P Cagg promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and Materials and Methods ). H, approximate locations of HindIII sites used in Southern analyses with DNA probe A (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe D (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes A and D , respectively. (G) Southern hybridization analysis using DNA probe A and HindIII-digested genomic DNA. The wt Ate1 allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed [10] unconditionally null Ate1 − allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on ( Ate1 flox ) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, Ate1 +/+ ; lane 2, Ate1 +/− ; lane 3, Ate1 +/− ; lane 4, Ate1 flox/− . (H) Southern hybridization analysis using DNA probe D (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed [10] unconditionally null Ate1 − allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type Ate1 allele and the flox-on ( Ate1 flox ) allele yield the 9.7 kB fragment, whereas the null flox-off ( Ate1 − ) allele yields the characteristic 3.8 kb fragment. The use of DNA probe D and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated Ate1 flox/− ; CaggCreER mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off ( Ate1 − ) allele. For example, whereas no flox-on ( Ate1 flox ) allele could be detected in the kidney and brain of Ate1 flox/− ; CaggCreER mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on ( Ate1 flox ) and flox-off ( Ate1 − ) alleles were present in the heart of TM-treated Ate1 flox/− ; CaggCreER mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed [10] Ate1 +/− mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated Ate1 flox/− ; CaggCreER mice. Lane 8, same as lane 7, but from a TM-treated Ate1 flox/− mouse (lacking the CaggCreER transgene).

    Journal: PLoS ONE

    Article Title: Ablation of Arginylation in the Mouse N-End Rule Pathway: Loss of Fat, Higher Metabolic Rate, Damaged Spermatogenesis, and Neurological Perturbations

    doi: 10.1371/journal.pone.0007757

    Figure Lengend Snippet: Genomic configurations at the Ate1 locus of Cre-lox -based mouse strains constructed in the present work. (A) The 5′ end of the previously produced unconditional Ate1 − allele [10] , in which the Ate1 exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P Ate1 promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P PGK promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse Ate1 , indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of Ate1 homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of Ate1 exon 2, a “floxed”-hygromycin-resistance ( hph ) cassette, expressed from the P PGK promoter (blue arrow between two red triangles) downstream of Ate1 exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P HSV promoter (yellow arrow). Wavy line indicates an abutting sequence of the pBR322 plasmid DNA. (D) The tri-lox Ate1 allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active Ate1 flox allele), whereas “flox-off” indicates a configuration depicted in panel F (the null Ate1 − allele). The functionally active, “flox-on” ( Ate1 flox ) allele, obtained by the removal of the hph cassette, using the in vivo expression of Cre-recombinase driven by the P EIIA promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” ( Ate1 − ) allele obtained by the inducible expression of CreER recombinase from the P Cagg promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and Materials and Methods ). H, approximate locations of HindIII sites used in Southern analyses with DNA probe A (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe D (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes A and D , respectively. (G) Southern hybridization analysis using DNA probe A and HindIII-digested genomic DNA. The wt Ate1 allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed [10] unconditionally null Ate1 − allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on ( Ate1 flox ) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, Ate1 +/+ ; lane 2, Ate1 +/− ; lane 3, Ate1 +/− ; lane 4, Ate1 flox/− . (H) Southern hybridization analysis using DNA probe D (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed [10] unconditionally null Ate1 − allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type Ate1 allele and the flox-on ( Ate1 flox ) allele yield the 9.7 kB fragment, whereas the null flox-off ( Ate1 − ) allele yields the characteristic 3.8 kb fragment. The use of DNA probe D and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated Ate1 flox/− ; CaggCreER mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off ( Ate1 − ) allele. For example, whereas no flox-on ( Ate1 flox ) allele could be detected in the kidney and brain of Ate1 flox/− ; CaggCreER mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on ( Ate1 flox ) and flox-off ( Ate1 − ) alleles were present in the heart of TM-treated Ate1 flox/− ; CaggCreER mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed [10] Ate1 +/− mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated Ate1 flox/− ; CaggCreER mice. Lane 8, same as lane 7, but from a TM-treated Ate1 flox/− mouse (lacking the CaggCreER transgene).

    Article Snippet: The entire ∼12 kb HindIII fragment and a part of the ∼2.9 kb fragment were modified as described below and assembled into a final ∼22.5 kb targeting vector consisting of the following parts ( ): ( i ) pBR322 backbone (New England Biolabs, Ipswich, MA); ( ii ) a ∼6.3 kb “long arm” of Ate1 homology containing the Ate1 exon 1a, the bidirectional PAte1 promoter , and exon 1b; ( iii ) A single loxP site ∼300 bp upstream of Ate1 exon 2; ( iv ) a ∼2 kb fragment that contains, 50 bp downstream of Ate1 exon 4, a “floxed” Hph (hygromycin) antibiotic-resistance marker, expressed from the PPGK promoter ; ( v ) a ∼1.2 kb “short arm” of Ate1 homology that spans most of the intron between exons 4 and 5; ( vi ) a gene encoding HSV-TK (herpes simplex virus thymidine kinase), expressed from the PPGK promoter.

    Techniques: Construct, Produced, Selection, Marker, Sequencing, Plasmid Preparation, In Vivo, Expressing, Hybridization, Mouse Assay