3-kb genomic dna fragment Search Results


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  • 99
    Zymo Research genomic dna
    Genomic Dna, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 11817 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs genomic dna
    Genomic Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 10746 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher genomic dna gdna
    Correction of the Z mutation in <t>A1ATD-hIPSCs</t> a , The strategy for precise genome modification using ZFNs and the piggyBac transposon. Top line, structure of the A1AT gene; blue lines, Southern blot probes; thin and thick boxes, non-coding and coding exons, respectively; open arrow, piggyBac transposon; B, Bam HI; A, Afl III. b , Sequences of wild-type (Reference), Z, PB , and Rev alleles. Amino acid position 342 (blue), recognition sites for ZFNs (green), piggyBac excision site (red) are shown. Sequence changes in Rev allele from Z allele were indicated by asterisks. c , Surveyor nuclease assay showing the cleavage of Z mutation in ZFNs-transfected K562 cells. Non-transfected cells were used as a control. d , Southern blot analysis showing bi-allelic piggyBac insertion (B-16) and bi-allelic excision (B-16-C2, -C3 and -C6) during correction of the A1ATD-hIPSCs line B. Genomic <t>DNA</t> was digested by Bam HI (5′ and PB probes) or Alf III (3′ probe). Genotype: ZZ, homozygous for Z allele; PP, homozygous for insertion of piggyBac ; RR, homozygous for reverted allele. e , Sequence analysis showing correction of Z mutation in 3 corrected hIPSC lines. Wild-type sequence (top line) and A1ATD-hIPSC (second line).
    Genomic Dna Gdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher chargeswitch gdna kit
    Correction of the Z mutation in <t>A1ATD-hIPSCs</t> a , The strategy for precise genome modification using ZFNs and the piggyBac transposon. Top line, structure of the A1AT gene; blue lines, Southern blot probes; thin and thick boxes, non-coding and coding exons, respectively; open arrow, piggyBac transposon; B, Bam HI; A, Afl III. b , Sequences of wild-type (Reference), Z, PB , and Rev alleles. Amino acid position 342 (blue), recognition sites for ZFNs (green), piggyBac excision site (red) are shown. Sequence changes in Rev allele from Z allele were indicated by asterisks. c , Surveyor nuclease assay showing the cleavage of Z mutation in ZFNs-transfected K562 cells. Non-transfected cells were used as a control. d , Southern blot analysis showing bi-allelic piggyBac insertion (B-16) and bi-allelic excision (B-16-C2, -C3 and -C6) during correction of the A1ATD-hIPSCs line B. Genomic <t>DNA</t> was digested by Bam HI (5′ and PB probes) or Alf III (3′ probe). Genotype: ZZ, homozygous for Z allele; PP, homozygous for insertion of piggyBac ; RR, homozygous for reverted allele. e , Sequence analysis showing correction of Z mutation in 3 corrected hIPSC lines. Wild-type sequence (top line) and A1ATD-hIPSC (second line).
    Chargeswitch Gdna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Iogen iogen m4 gdna
    Correction of the Z mutation in <t>A1ATD-hIPSCs</t> a , The strategy for precise genome modification using ZFNs and the piggyBac transposon. Top line, structure of the A1AT gene; blue lines, Southern blot probes; thin and thick boxes, non-coding and coding exons, respectively; open arrow, piggyBac transposon; B, Bam HI; A, Afl III. b , Sequences of wild-type (Reference), Z, PB , and Rev alleles. Amino acid position 342 (blue), recognition sites for ZFNs (green), piggyBac excision site (red) are shown. Sequence changes in Rev allele from Z allele were indicated by asterisks. c , Surveyor nuclease assay showing the cleavage of Z mutation in ZFNs-transfected K562 cells. Non-transfected cells were used as a control. d , Southern blot analysis showing bi-allelic piggyBac insertion (B-16) and bi-allelic excision (B-16-C2, -C3 and -C6) during correction of the A1ATD-hIPSCs line B. Genomic <t>DNA</t> was digested by Bam HI (5′ and PB probes) or Alf III (3′ probe). Genotype: ZZ, homozygous for Z allele; PP, homozygous for insertion of piggyBac ; RR, homozygous for reverted allele. e , Sequence analysis showing correction of Z mutation in 3 corrected hIPSC lines. Wild-type sequence (top line) and A1ATD-hIPSC (second line).
    Iogen M4 Gdna, supplied by Iogen, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Iogen iogen m8 gdna
    Correction of the Z mutation in <t>A1ATD-hIPSCs</t> a , The strategy for precise genome modification using ZFNs and the piggyBac transposon. Top line, structure of the A1AT gene; blue lines, Southern blot probes; thin and thick boxes, non-coding and coding exons, respectively; open arrow, piggyBac transposon; B, Bam HI; A, Afl III. b , Sequences of wild-type (Reference), Z, PB , and Rev alleles. Amino acid position 342 (blue), recognition sites for ZFNs (green), piggyBac excision site (red) are shown. Sequence changes in Rev allele from Z allele were indicated by asterisks. c , Surveyor nuclease assay showing the cleavage of Z mutation in ZFNs-transfected K562 cells. Non-transfected cells were used as a control. d , Southern blot analysis showing bi-allelic piggyBac insertion (B-16) and bi-allelic excision (B-16-C2, -C3 and -C6) during correction of the A1ATD-hIPSCs line B. Genomic <t>DNA</t> was digested by Bam HI (5′ and PB probes) or Alf III (3′ probe). Genotype: ZZ, homozygous for Z allele; PP, homozygous for insertion of piggyBac ; RR, homozygous for reverted allele. e , Sequence analysis showing correction of Z mutation in 3 corrected hIPSC lines. Wild-type sequence (top line) and A1ATD-hIPSC (second line).
    Iogen M8 Gdna, supplied by Iogen, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATUM genomic dna
    Correction of the Z mutation in <t>A1ATD-hIPSCs</t> a , The strategy for precise genome modification using ZFNs and the piggyBac transposon. Top line, structure of the A1AT gene; blue lines, Southern blot probes; thin and thick boxes, non-coding and coding exons, respectively; open arrow, piggyBac transposon; B, Bam HI; A, Afl III. b , Sequences of wild-type (Reference), Z, PB , and Rev alleles. Amino acid position 342 (blue), recognition sites for ZFNs (green), piggyBac excision site (red) are shown. Sequence changes in Rev allele from Z allele were indicated by asterisks. c , Surveyor nuclease assay showing the cleavage of Z mutation in ZFNs-transfected K562 cells. Non-transfected cells were used as a control. d , Southern blot analysis showing bi-allelic piggyBac insertion (B-16) and bi-allelic excision (B-16-C2, -C3 and -C6) during correction of the A1ATD-hIPSCs line B. Genomic <t>DNA</t> was digested by Bam HI (5′ and PB probes) or Alf III (3′ probe). Genotype: ZZ, homozygous for Z allele; PP, homozygous for insertion of piggyBac ; RR, homozygous for reverted allele. e , Sequence analysis showing correction of Z mutation in 3 corrected hIPSC lines. Wild-type sequence (top line) and A1ATD-hIPSC (second line).
    Genomic Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 94/100, based on 2613 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore kb genomic dna fragment
    Disruption of the FOR3 gene in F. <t>oxysporum</t> f. sp. nicotianae . (A) Schematic view of the FOR3 gene disruption strategy. The <t>DNA</t> inserts of p FOR3 and p for3 ::HYG are shown. The FOR3 coding region is indicated by the gray striped box and noncoding regions by the gray line. The vector sequences are indicated by the black line. Restriction sites on the cloning vector are indicated in black, and those in FOR3 are indicated in gray. The sizes of cloned DNA fragments (double-headed arrows), as well as the probes used for Southern (dotted line) or Northern (dashed line) blot analyses, are indicated. Restriction site abbreviations are as follows: E, EcoRV; E1, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SalI; Xb, XbaI; Xh, XhoI. (B) Southern blot of restriction-digested genomic DNA of the wild-type ( FOR3 ) and Δ for3 mutant strains. Restriction site abbreviations are as in panel A. (C) Northern blot of total RNA of the wild-type ( FOR3 ) and Δ for3 mutant strains with a FOR3 probe ( FOR3 ) and the corresponding ethidium bromide signal (RNA) shown.
    Kb Genomic Dna Fragment, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen genomic dna
    Expression of genes surrounding the hCD3ε transgenic integration site in tgε26 +/+ thymocytes. (A) Quantitative <t>PCR</t> analysis of mRNA expression in neonatal tgε26 +/− (open bars) and tgε26 +/+ (black bars) thymocytes. Results were normalized to GAPDH mRNA and quantitated relative to DN1 cells isolated from WT mice. The expression of Tpsb2 , Gng13 , Msln , Ccdc78 and Pdia2 in tgε26 +/+ thymocytes (gray bars) was quantitated relative to tgε26 +/− thymocytes, because expression of these genes was not detected in WT thymocytes. Means and standard errors of 3 independent measurements are shown. nd: not detected. Primer sequences are shown in Supplementary Table S4 . (B) RNA isolated from thymocytes (Thy), splenocytes (Spl), bone marrow cells (BM), and genomic <t>DNA</t> (gDNA) of WT mice was electrophoresed on a polyacrylamide gel and then blotted and probed for TS1F/TS1R sequence ( Fig. 2E ), m7900/m8100 sequence ( Fig. 2H ) and U6 small nuclear RNA. Representative results from two independent experiments are shown.
    Genomic Dna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 127055 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa mouse genomic dna
    Time-course <t>DNA</t> microarray analysis of 3T3-L1 cells treated with MDI plus DMSO or MDI plus echinomycin. ( A ) Clustering analysis performed among the MDI + DMSO treated groups using 2,204 selected probes that had a fold change ≥ 2.0 in expression compared with the control (0 hr) (See the details in Methods). Genes were clustered into 3 groups according to their timing of response to MDI treatment. ( B ) (a,b) Scatter plot of all the 14,005 probes (signal intensity ≥ 100 at any one point) at 2 hr and 6 hr. A log2-fold change from the control (time 0 hr) of the MDI + echinomycin group is plotted against a log2-fold change from the control (time 0 hr) of the MDI + DMSO group. (c) The scheme of probe selection for further clustering. Genes that had a fold change ≥ 2.0 (x ≥ 1) at both 2 hr and 6 hr relative to time 0 preadipocytes were further analysed using hierarchical clustering. ( C ) Representative quantitative <t>RT-PCR</t> analysis of Cebpb , Cebpd , Klf4 , and Klf5 were performed to validate the microarray analysis. The data are the mean ± SEM of three independent experiments. *P
    Mouse Genomic Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 691 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega mouse genomic dna fragment
    The effect of PPARδ activation on ischemia-induced cerebrovascular permeability and potential mechanisms in vivo . Mice were pretreated with a specific PPARδ agonist, GW 501516, or vehicle reagent for 24h via mini-pump assisted-cerebroventricular infusion, and then subjected to 1h MCA occlusion and 24h-reperfusion. 2% TTC-stained coronal sections were shown at different brain levels posterior to the frontal pole (A). Quantitative analysis was made on brain infarct volume (B), and cerebrovascular permeability was determined 1h after injection from Evans Blue extravasation as described in the Methods (C) (n=6) in mice after stroke. In comparison to the vehicle control, treatment of GW 501516 significantly attenuates ischemic brain infarction and improves cerebrovascular permeability. ICV infusion of GW 501516 also significantly reduced ischemia-induced <t>miR-15a</t> upregulation (D), increased bcl-2 protein levels (E), and attenuated caspase-3 activation (F) and <t>DNA</t> fragmentation (G) in the cerebral microvasculature in mice after MCAO. Data are expressed as mean ± SD. * p
    Mouse Genomic Dna Fragment, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PerkinElmer genomic dna
    The effect of PPARδ activation on ischemia-induced cerebrovascular permeability and potential mechanisms in vivo . Mice were pretreated with a specific PPARδ agonist, GW 501516, or vehicle reagent for 24h via mini-pump assisted-cerebroventricular infusion, and then subjected to 1h MCA occlusion and 24h-reperfusion. 2% TTC-stained coronal sections were shown at different brain levels posterior to the frontal pole (A). Quantitative analysis was made on brain infarct volume (B), and cerebrovascular permeability was determined 1h after injection from Evans Blue extravasation as described in the Methods (C) (n=6) in mice after stroke. In comparison to the vehicle control, treatment of GW 501516 significantly attenuates ischemic brain infarction and improves cerebrovascular permeability. ICV infusion of GW 501516 also significantly reduced ischemia-induced <t>miR-15a</t> upregulation (D), increased bcl-2 protein levels (E), and attenuated caspase-3 activation (F) and <t>DNA</t> fragmentation (G) in the cerebral microvasculature in mice after MCAO. Data are expressed as mean ± SD. * p
    Genomic Dna, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 97/100, based on 1090 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore genomic dna
    Alignment of JSRV <t>JS7</t> provirus restriction maps. (A) Results of Southern blot analysis of JS7 genomic <t>DNA</t> digested with restriction endonucleases Eco RI (E), Hin dIII (H), and Sac ). (B) Full-length proviral lambda clone 2-1. (C) Partial-length proviral lambda clone 5-1.
    Genomic Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10774 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche genomic dna
    Alignment of JSRV <t>JS7</t> provirus restriction maps. (A) Results of Southern blot analysis of JS7 genomic <t>DNA</t> digested with restriction endonucleases Eco RI (E), Hin dIII (H), and Sac ). (B) Full-length proviral lambda clone 2-1. (C) Partial-length proviral lambda clone 5-1.
    Genomic Dna, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 10355 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pacific Biosciences bp 3 kb fragments
    Alignment of JSRV <t>JS7</t> provirus restriction maps. (A) Results of Southern blot analysis of JS7 genomic <t>DNA</t> digested with restriction endonucleases Eco RI (E), Hin dIII (H), and Sac ). (B) Full-length proviral lambda clone 2-1. (C) Partial-length proviral lambda clone 5-1.
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    Millipore genelute bacterial genomic dna kit
    Alignment of JSRV <t>JS7</t> provirus restriction maps. (A) Results of Southern blot analysis of JS7 genomic <t>DNA</t> digested with restriction endonucleases Eco RI (E), Hin dIII (H), and Sac ). (B) Full-length proviral lambda clone 2-1. (C) Partial-length proviral lambda clone 5-1.
    Genelute Bacterial Genomic Dna Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3476 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega genomic dna purification kit
    Alignment of JSRV <t>JS7</t> provirus restriction maps. (A) Results of Southern blot analysis of JS7 genomic <t>DNA</t> digested with restriction endonucleases Eco RI (E), Hin dIII (H), and Sac ). (B) Full-length proviral lambda clone 2-1. (C) Partial-length proviral lambda clone 5-1.
    Genomic Dna Purification Kit, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 1902 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Correction of the Z mutation in A1ATD-hIPSCs a , The strategy for precise genome modification using ZFNs and the piggyBac transposon. Top line, structure of the A1AT gene; blue lines, Southern blot probes; thin and thick boxes, non-coding and coding exons, respectively; open arrow, piggyBac transposon; B, Bam HI; A, Afl III. b , Sequences of wild-type (Reference), Z, PB , and Rev alleles. Amino acid position 342 (blue), recognition sites for ZFNs (green), piggyBac excision site (red) are shown. Sequence changes in Rev allele from Z allele were indicated by asterisks. c , Surveyor nuclease assay showing the cleavage of Z mutation in ZFNs-transfected K562 cells. Non-transfected cells were used as a control. d , Southern blot analysis showing bi-allelic piggyBac insertion (B-16) and bi-allelic excision (B-16-C2, -C3 and -C6) during correction of the A1ATD-hIPSCs line B. Genomic DNA was digested by Bam HI (5′ and PB probes) or Alf III (3′ probe). Genotype: ZZ, homozygous for Z allele; PP, homozygous for insertion of piggyBac ; RR, homozygous for reverted allele. e , Sequence analysis showing correction of Z mutation in 3 corrected hIPSC lines. Wild-type sequence (top line) and A1ATD-hIPSC (second line).

    Journal: Nature

    Article Title: Targeted gene correction of ?1-antitrypsin deficiency in induced pluripotent stem cells

    doi: 10.1038/nature10424

    Figure Lengend Snippet: Correction of the Z mutation in A1ATD-hIPSCs a , The strategy for precise genome modification using ZFNs and the piggyBac transposon. Top line, structure of the A1AT gene; blue lines, Southern blot probes; thin and thick boxes, non-coding and coding exons, respectively; open arrow, piggyBac transposon; B, Bam HI; A, Afl III. b , Sequences of wild-type (Reference), Z, PB , and Rev alleles. Amino acid position 342 (blue), recognition sites for ZFNs (green), piggyBac excision site (red) are shown. Sequence changes in Rev allele from Z allele were indicated by asterisks. c , Surveyor nuclease assay showing the cleavage of Z mutation in ZFNs-transfected K562 cells. Non-transfected cells were used as a control. d , Southern blot analysis showing bi-allelic piggyBac insertion (B-16) and bi-allelic excision (B-16-C2, -C3 and -C6) during correction of the A1ATD-hIPSCs line B. Genomic DNA was digested by Bam HI (5′ and PB probes) or Alf III (3′ probe). Genotype: ZZ, homozygous for Z allele; PP, homozygous for insertion of piggyBac ; RR, homozygous for reverted allele. e , Sequence analysis showing correction of Z mutation in 3 corrected hIPSC lines. Wild-type sequence (top line) and A1ATD-hIPSC (second line).

    Article Snippet: A donor template vector for A1AT : A 2-kb fragment, which contained 1 kb at both side of Z mutation, was first PCR-amplified using genomic DNA from A1ATD-hIPSC line B as a template and cloned into pCR4-blunt-TOPO (Invitrogen), resulting in pCR4-AAT_Z.

    Techniques: Mutagenesis, Modification, Southern Blot, Sequencing, Nuclease Assay, Transfection

    Functional analysis of restored A1AT in c-hIPSCs-derived hepatocyte-like cells a , Immunofluorescence showing the absence of polymeric A1AT protein in hepatocyte-like cells generated from c-hIPSCs. All forms of A1AT (left panels) and misfolded polymeric A1AT (middle panels). b , c , ELISA to assess the intracellular ( b ) and secreted ( c ) levels of polymeric A1AT protein in hepatocyte-like cells derived from A1ATD-hIPSCs (ZZ), c-hIPSCs (RR) and control hIPSCs (++). d , Endoglycosidase H (E) and peptide: N -glycosidase (P) digestion of A1AT immunoprecipitated from uncorrected (ZZ), corrected (RR) and control (++) hIPSC-derived hepatocyte-like cells (upper panels) and corresponding culture medium (lower panels). e , Chymotrypsin ELISA showing that corrected cells (RR) have A1AT enzymatic inhibitory activity that is superior to uncorrected cells (ZZ) and close to adult hepatocytes. f , g , Immunofluorescence of transplanted liver sections detecting human albumin ( f ) and A1AT ( g ). DNA was counterstained with DAPI. h , ELISA read-out of human albumin in the mouse serum longitudinally followed for each mouse. Asterisk, the mouse was subjected to histology analysis. Scale bars, 100 μm. Data in b , c and e are shown as mean ± s.d. ( n =3). Student’s t -test was performed. NS, not significant.

    Journal: Nature

    Article Title: Targeted gene correction of ?1-antitrypsin deficiency in induced pluripotent stem cells

    doi: 10.1038/nature10424

    Figure Lengend Snippet: Functional analysis of restored A1AT in c-hIPSCs-derived hepatocyte-like cells a , Immunofluorescence showing the absence of polymeric A1AT protein in hepatocyte-like cells generated from c-hIPSCs. All forms of A1AT (left panels) and misfolded polymeric A1AT (middle panels). b , c , ELISA to assess the intracellular ( b ) and secreted ( c ) levels of polymeric A1AT protein in hepatocyte-like cells derived from A1ATD-hIPSCs (ZZ), c-hIPSCs (RR) and control hIPSCs (++). d , Endoglycosidase H (E) and peptide: N -glycosidase (P) digestion of A1AT immunoprecipitated from uncorrected (ZZ), corrected (RR) and control (++) hIPSC-derived hepatocyte-like cells (upper panels) and corresponding culture medium (lower panels). e , Chymotrypsin ELISA showing that corrected cells (RR) have A1AT enzymatic inhibitory activity that is superior to uncorrected cells (ZZ) and close to adult hepatocytes. f , g , Immunofluorescence of transplanted liver sections detecting human albumin ( f ) and A1AT ( g ). DNA was counterstained with DAPI. h , ELISA read-out of human albumin in the mouse serum longitudinally followed for each mouse. Asterisk, the mouse was subjected to histology analysis. Scale bars, 100 μm. Data in b , c and e are shown as mean ± s.d. ( n =3). Student’s t -test was performed. NS, not significant.

    Article Snippet: A donor template vector for A1AT : A 2-kb fragment, which contained 1 kb at both side of Z mutation, was first PCR-amplified using genomic DNA from A1ATD-hIPSC line B as a template and cloned into pCR4-blunt-TOPO (Invitrogen), resulting in pCR4-AAT_Z.

    Techniques: Functional Assay, Derivative Assay, Immunofluorescence, Generated, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Activity Assay

    Disruption of the FOR3 gene in F. oxysporum f. sp. nicotianae . (A) Schematic view of the FOR3 gene disruption strategy. The DNA inserts of p FOR3 and p for3 ::HYG are shown. The FOR3 coding region is indicated by the gray striped box and noncoding regions by the gray line. The vector sequences are indicated by the black line. Restriction sites on the cloning vector are indicated in black, and those in FOR3 are indicated in gray. The sizes of cloned DNA fragments (double-headed arrows), as well as the probes used for Southern (dotted line) or Northern (dashed line) blot analyses, are indicated. Restriction site abbreviations are as follows: E, EcoRV; E1, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SalI; Xb, XbaI; Xh, XhoI. (B) Southern blot of restriction-digested genomic DNA of the wild-type ( FOR3 ) and Δ for3 mutant strains. Restriction site abbreviations are as in panel A. (C) Northern blot of total RNA of the wild-type ( FOR3 ) and Δ for3 mutant strains with a FOR3 probe ( FOR3 ) and the corresponding ethidium bromide signal (RNA) shown.

    Journal: Eukaryotic Cell

    Article Title: Use of the Plant Defense Protein Osmotin To Identify Fusarium oxysporum Genes That Control Cell Wall Properties ▿ Genes That Control Cell Wall Properties ▿ ‡

    doi: 10.1128/EC.00316-09

    Figure Lengend Snippet: Disruption of the FOR3 gene in F. oxysporum f. sp. nicotianae . (A) Schematic view of the FOR3 gene disruption strategy. The DNA inserts of p FOR3 and p for3 ::HYG are shown. The FOR3 coding region is indicated by the gray striped box and noncoding regions by the gray line. The vector sequences are indicated by the black line. Restriction sites on the cloning vector are indicated in black, and those in FOR3 are indicated in gray. The sizes of cloned DNA fragments (double-headed arrows), as well as the probes used for Southern (dotted line) or Northern (dashed line) blot analyses, are indicated. Restriction site abbreviations are as follows: E, EcoRV; E1, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SalI; Xb, XbaI; Xh, XhoI. (B) Southern blot of restriction-digested genomic DNA of the wild-type ( FOR3 ) and Δ for3 mutant strains. Restriction site abbreviations are as in panel A. (C) Northern blot of total RNA of the wild-type ( FOR3 ) and Δ for3 mutant strains with a FOR3 probe ( FOR3 ) and the corresponding ethidium bromide signal (RNA) shown.

    Article Snippet: To construct p for3 ::HYG, a 0.7-kb fragment containing the 3′ part of the FOR3 ORF (+3205 bp to +3941 bp) was amplified from F. oxysporum genomic DNA by PCR using the primer pair 5′-ATCGAGTCTTGCCGACGAT-3′/5′-TGAGATCCGTCTTCAGGATC-3′ and inserted into the EcoRV site of the pSTblue-1 vector (Novagen, Madison, WI).

    Techniques: Plasmid Preparation, Clone Assay, Northern Blot, Southern Blot, Mutagenesis

    FOR1 , FOR2 , and FOR3 increase osmotin resistance of S. cerevisiae cells and are single-copy genes in F. oxysporum f. sp. nicotianae . (A, B, and C) Shown are osmotin sensitivities of cells of S. cerevisiae strain BWG1-7a transformed with p416GPD (vector) or the cDNA clone p FOR1 (A), p FOR2 (B), or p FOR3 (C), which were compared by estimating growth in the presence of various concentrations of osmotin. Growth of cells was measured in liquid culture in selective minimal medium supplemented with the indicated osmotin concentrations. Data were normalized to viable counts of samples without osmotin. Data are the averages ± standard errors (SE) of results from at least three experiments with duplicate samples. (D) Shown are blots of restriction enzyme-digested genomic DNA (15 μg) probed with 32 P-labeled FOR1 (lanes 1, 2, and 3), FOR2 (lanes 4, 5, and 6), and FOR3 (lanes 7, 8, and 9). The restriction enzymes used were EcoRI (lanes 1, 4, and 7), PstI (lanes 2, 5, and 8), and XbaI (lanes 3, 6, and 9).

    Journal: Eukaryotic Cell

    Article Title: Use of the Plant Defense Protein Osmotin To Identify Fusarium oxysporum Genes That Control Cell Wall Properties ▿ Genes That Control Cell Wall Properties ▿ ‡

    doi: 10.1128/EC.00316-09

    Figure Lengend Snippet: FOR1 , FOR2 , and FOR3 increase osmotin resistance of S. cerevisiae cells and are single-copy genes in F. oxysporum f. sp. nicotianae . (A, B, and C) Shown are osmotin sensitivities of cells of S. cerevisiae strain BWG1-7a transformed with p416GPD (vector) or the cDNA clone p FOR1 (A), p FOR2 (B), or p FOR3 (C), which were compared by estimating growth in the presence of various concentrations of osmotin. Growth of cells was measured in liquid culture in selective minimal medium supplemented with the indicated osmotin concentrations. Data were normalized to viable counts of samples without osmotin. Data are the averages ± standard errors (SE) of results from at least three experiments with duplicate samples. (D) Shown are blots of restriction enzyme-digested genomic DNA (15 μg) probed with 32 P-labeled FOR1 (lanes 1, 2, and 3), FOR2 (lanes 4, 5, and 6), and FOR3 (lanes 7, 8, and 9). The restriction enzymes used were EcoRI (lanes 1, 4, and 7), PstI (lanes 2, 5, and 8), and XbaI (lanes 3, 6, and 9).

    Article Snippet: To construct p for3 ::HYG, a 0.7-kb fragment containing the 3′ part of the FOR3 ORF (+3205 bp to +3941 bp) was amplified from F. oxysporum genomic DNA by PCR using the primer pair 5′-ATCGAGTCTTGCCGACGAT-3′/5′-TGAGATCCGTCTTCAGGATC-3′ and inserted into the EcoRV site of the pSTblue-1 vector (Novagen, Madison, WI).

    Techniques: Transformation Assay, Plasmid Preparation, Labeling

    Protective capacity of individual antigens against sporozoite challenge. Mice (n = 5–15/group representative of up to three independent experiments; see Table 3 ) were immunised with (I) plasmid DNA/recombinant protein expressing the target antigens or (II) pools of synthetic peptides representing predicted CD8 + and CD4 + T cell epitopes for each antigen formulated in AbISCO100 adjuvant. Controls were immunised with empty vector only (I), or adjuvant only (II), or not immunised (dotted line) . Mice were challenged with 10 3 P. yoelii cryopreserved infectious sporozoites or 10 3 P. yoelii pRBC at 14 days post last immunisation. Protection was assessed by (A) qRT-PCR of Py18S rRNA in total liver RNA at 42 h after sporozoite challenge normalised against the 18S rRNA of infected but not vaccinated mice (dotted line); and (B) FCAB assay for days 3 to 30 after blood-stage challenge normalised against AUC of infectivity controls (dotted line) . Data are presented as a scatter plot, with the line representing the mean and the error bars representing standard deviation (SD). Statistical comparison of immunised versus non-immunised infectivity controls was performed using one-way ANOVA followed by Bonferroni’s posthoc test, **** p

    Journal: Scientific Reports

    Article Title: Novel Plasmodium antigens identified via genome-based antibody screen induce protection associated with polyfunctional T cell responses

    doi: 10.1038/s41598-017-15354-0

    Figure Lengend Snippet: Protective capacity of individual antigens against sporozoite challenge. Mice (n = 5–15/group representative of up to three independent experiments; see Table 3 ) were immunised with (I) plasmid DNA/recombinant protein expressing the target antigens or (II) pools of synthetic peptides representing predicted CD8 + and CD4 + T cell epitopes for each antigen formulated in AbISCO100 adjuvant. Controls were immunised with empty vector only (I), or adjuvant only (II), or not immunised (dotted line) . Mice were challenged with 10 3 P. yoelii cryopreserved infectious sporozoites or 10 3 P. yoelii pRBC at 14 days post last immunisation. Protection was assessed by (A) qRT-PCR of Py18S rRNA in total liver RNA at 42 h after sporozoite challenge normalised against the 18S rRNA of infected but not vaccinated mice (dotted line); and (B) FCAB assay for days 3 to 30 after blood-stage challenge normalised against AUC of infectivity controls (dotted line) . Data are presented as a scatter plot, with the line representing the mean and the error bars representing standard deviation (SD). Statistical comparison of immunised versus non-immunised infectivity controls was performed using one-way ANOVA followed by Bonferroni’s posthoc test, **** p

    Article Snippet: Amplification of each target gene from P. yoelii genomic DNA was conducted in a 50-μl PCR reaction containing 1 unit/μl KOD Hot Start DNA polymerase in corresponding buffer (Novagen, San Diego, CA), 0.4 μM dNTPs, 333 nM of each primer and 100 ng DNA template.

    Techniques: Mouse Assay, Plasmid Preparation, Recombinant, Expressing, Quantitative RT-PCR, Infection, Standard Deviation

    Expression of genes surrounding the hCD3ε transgenic integration site in tgε26 +/+ thymocytes. (A) Quantitative PCR analysis of mRNA expression in neonatal tgε26 +/− (open bars) and tgε26 +/+ (black bars) thymocytes. Results were normalized to GAPDH mRNA and quantitated relative to DN1 cells isolated from WT mice. The expression of Tpsb2 , Gng13 , Msln , Ccdc78 and Pdia2 in tgε26 +/+ thymocytes (gray bars) was quantitated relative to tgε26 +/− thymocytes, because expression of these genes was not detected in WT thymocytes. Means and standard errors of 3 independent measurements are shown. nd: not detected. Primer sequences are shown in Supplementary Table S4 . (B) RNA isolated from thymocytes (Thy), splenocytes (Spl), bone marrow cells (BM), and genomic DNA (gDNA) of WT mice was electrophoresed on a polyacrylamide gel and then blotted and probed for TS1F/TS1R sequence ( Fig. 2E ), m7900/m8100 sequence ( Fig. 2H ) and U6 small nuclear RNA. Representative results from two independent experiments are shown.

    Journal: PLoS ONE

    Article Title: Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice

    doi: 10.1371/journal.pone.0014391

    Figure Lengend Snippet: Expression of genes surrounding the hCD3ε transgenic integration site in tgε26 +/+ thymocytes. (A) Quantitative PCR analysis of mRNA expression in neonatal tgε26 +/− (open bars) and tgε26 +/+ (black bars) thymocytes. Results were normalized to GAPDH mRNA and quantitated relative to DN1 cells isolated from WT mice. The expression of Tpsb2 , Gng13 , Msln , Ccdc78 and Pdia2 in tgε26 +/+ thymocytes (gray bars) was quantitated relative to tgε26 +/− thymocytes, because expression of these genes was not detected in WT thymocytes. Means and standard errors of 3 independent measurements are shown. nd: not detected. Primer sequences are shown in Supplementary Table S4 . (B) RNA isolated from thymocytes (Thy), splenocytes (Spl), bone marrow cells (BM), and genomic DNA (gDNA) of WT mice was electrophoresed on a polyacrylamide gel and then blotted and probed for TS1F/TS1R sequence ( Fig. 2E ), m7900/m8100 sequence ( Fig. 2H ) and U6 small nuclear RNA. Representative results from two independent experiments are shown.

    Article Snippet: For inverse PCR, tgε26+/+ genomic DNA was digested with NcoI, and 2–3 kb DNA fragments were extracted from agarose gel using the QIAquick Gel Extraction Kit (Qiagen) and were self-ligated using T4 DNA ligase (Takara) and PCR amplified using the i-1F ( 5′-TATCCGAGCCAAATGTGCCA-3′ ) and i-1R ( 5′-AGATAGAAACCTCAATGCCCA-3′ ) primers.

    Techniques: Expressing, Transgenic Assay, Real-time Polymerase Chain Reaction, Isolation, Mouse Assay, Sequencing

    Linkage analysis of chromosome 17 with T cell deficiency in tgε26 mice. Homozygous tgε26 mice were crossed with tgε26×C57BL/6 heterozygous mice. Peripheral blood cells from the offspring were 2-color stained for CD3 and TCRβ. (A) Genomic DNA from 30 CD3 + TCRβ + cell-positive mice and 29 CD3 + TCRβ + cell-negative mice was assessed for the indicated microsatellite markers. (B) Genomic DNA from 3 CD3 + TCRβ + cell-positive mice and 6 CD3 + TCRβ + cell-negative mice was assessed for the indicated microsatellite markers. C: CBA/J homozygous alleles, C/B: CBA/J and C57BL/6 heterozygous alleles, B: C57BL/6 homozygous alleles, -: not detected. Sequences of microsatellite markers located between D17mit26 and D17mit80 are listed in Supplementary Table S1 .

    Journal: PLoS ONE

    Article Title: Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice

    doi: 10.1371/journal.pone.0014391

    Figure Lengend Snippet: Linkage analysis of chromosome 17 with T cell deficiency in tgε26 mice. Homozygous tgε26 mice were crossed with tgε26×C57BL/6 heterozygous mice. Peripheral blood cells from the offspring were 2-color stained for CD3 and TCRβ. (A) Genomic DNA from 30 CD3 + TCRβ + cell-positive mice and 29 CD3 + TCRβ + cell-negative mice was assessed for the indicated microsatellite markers. (B) Genomic DNA from 3 CD3 + TCRβ + cell-positive mice and 6 CD3 + TCRβ + cell-negative mice was assessed for the indicated microsatellite markers. C: CBA/J homozygous alleles, C/B: CBA/J and C57BL/6 heterozygous alleles, B: C57BL/6 homozygous alleles, -: not detected. Sequences of microsatellite markers located between D17mit26 and D17mit80 are listed in Supplementary Table S1 .

    Article Snippet: For inverse PCR, tgε26+/+ genomic DNA was digested with NcoI, and 2–3 kb DNA fragments were extracted from agarose gel using the QIAquick Gel Extraction Kit (Qiagen) and were self-ligated using T4 DNA ligase (Takara) and PCR amplified using the i-1F ( 5′-TATCCGAGCCAAATGTGCCA-3′ ) and i-1R ( 5′-AGATAGAAACCTCAATGCCCA-3′ ) primers.

    Techniques: Mouse Assay, Staining, Crocin Bleaching Assay

    Identification of the tgε26 transgenic integration site. (A) Physical map of the genomic region between D17mit26-mit80-1 and D17mit 26-mit80-24. a–q: probes for Southern blot analysis. Details of these probes are listed in Supplementary 2 . 1–7: regions of quantitative genomic PCR analysis. S: SalI, E: EcoRI, A: AseI, K: KpnI, Hp: HpaI, Sc: SacI. (B–D) PFGE Southern blot analysis of SalI-digested WT and tgε26 +/+ genomic DNA using the indicated probes. (E) Sequence of Sstr5 -sequence-containing tgε26 +/+ genomic clones that also contained hCD3ε sequence (left). Physical map of the transgenic integration site at the Sstr5 locus (middle). H: HindIII. i: probe for Southern blot analysis. TS1F, TS1R, and CD3e9130F: primers for genomic PCR analysis. Genomic PCR analysis using TS1F/TS1R primers (lanes 1 and 2) and CDe9130F/TS1R primers (lanes 3 and 4) (right). (F, G) Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (H) Physical map of the transgenic integration site at the Metrn locus (left). N: NcoI. q: probe for Southern blot analysis. m7900, m8100, and CD3e23010F: primers for genomic PCR analysis. i-1R, i-1F, i-2R, and i-2F: primers for inverse and nested PCR amplification. Sequence of tgε26 +/+ genomic clones containing both Metrn and hCD3ε sequences (middle). Genomic PCR analysis using m7900/m8100 primers (lanes 1 and 2) and m7900/CD3e23010F primers (lanes 3 and 4) (right). (I, J) PFGE Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (K) Quantitative genomic PCR analysis of WT (open bars) and tgε26 +/+ (black bars) genomic DNA. Primers 1–7 are shown in (A). ct-1 and ct-2 are the genomic regions from chromosome 6 used as controls. Primer sequences are shown in Supplementary Table S3 . Data were normalized to ct-1 signals. Means and standard errors of 3 independent measurements are shown. (L) Configuration of the tgε26 allele.

    Journal: PLoS ONE

    Article Title: Identification of the Transgenic Integration Site in Immunodeficient tg?26 Human CD3? Transgenic Mice

    doi: 10.1371/journal.pone.0014391

    Figure Lengend Snippet: Identification of the tgε26 transgenic integration site. (A) Physical map of the genomic region between D17mit26-mit80-1 and D17mit 26-mit80-24. a–q: probes for Southern blot analysis. Details of these probes are listed in Supplementary 2 . 1–7: regions of quantitative genomic PCR analysis. S: SalI, E: EcoRI, A: AseI, K: KpnI, Hp: HpaI, Sc: SacI. (B–D) PFGE Southern blot analysis of SalI-digested WT and tgε26 +/+ genomic DNA using the indicated probes. (E) Sequence of Sstr5 -sequence-containing tgε26 +/+ genomic clones that also contained hCD3ε sequence (left). Physical map of the transgenic integration site at the Sstr5 locus (middle). H: HindIII. i: probe for Southern blot analysis. TS1F, TS1R, and CD3e9130F: primers for genomic PCR analysis. Genomic PCR analysis using TS1F/TS1R primers (lanes 1 and 2) and CDe9130F/TS1R primers (lanes 3 and 4) (right). (F, G) Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (H) Physical map of the transgenic integration site at the Metrn locus (left). N: NcoI. q: probe for Southern blot analysis. m7900, m8100, and CD3e23010F: primers for genomic PCR analysis. i-1R, i-1F, i-2R, and i-2F: primers for inverse and nested PCR amplification. Sequence of tgε26 +/+ genomic clones containing both Metrn and hCD3ε sequences (middle). Genomic PCR analysis using m7900/m8100 primers (lanes 1 and 2) and m7900/CD3e23010F primers (lanes 3 and 4) (right). (I, J) PFGE Southern blot analysis of WT and tgε26 +/+ genomic DNA digested with the indicated restriction enzymes using the indicated probes. (K) Quantitative genomic PCR analysis of WT (open bars) and tgε26 +/+ (black bars) genomic DNA. Primers 1–7 are shown in (A). ct-1 and ct-2 are the genomic regions from chromosome 6 used as controls. Primer sequences are shown in Supplementary Table S3 . Data were normalized to ct-1 signals. Means and standard errors of 3 independent measurements are shown. (L) Configuration of the tgε26 allele.

    Article Snippet: For inverse PCR, tgε26+/+ genomic DNA was digested with NcoI, and 2–3 kb DNA fragments were extracted from agarose gel using the QIAquick Gel Extraction Kit (Qiagen) and were self-ligated using T4 DNA ligase (Takara) and PCR amplified using the i-1F ( 5′-TATCCGAGCCAAATGTGCCA-3′ ) and i-1R ( 5′-AGATAGAAACCTCAATGCCCA-3′ ) primers.

    Techniques: Transgenic Assay, Southern Blot, Polymerase Chain Reaction, Sequencing, Clone Assay, Nested PCR, Amplification

    Time-course DNA microarray analysis of 3T3-L1 cells treated with MDI plus DMSO or MDI plus echinomycin. ( A ) Clustering analysis performed among the MDI + DMSO treated groups using 2,204 selected probes that had a fold change ≥ 2.0 in expression compared with the control (0 hr) (See the details in Methods). Genes were clustered into 3 groups according to their timing of response to MDI treatment. ( B ) (a,b) Scatter plot of all the 14,005 probes (signal intensity ≥ 100 at any one point) at 2 hr and 6 hr. A log2-fold change from the control (time 0 hr) of the MDI + echinomycin group is plotted against a log2-fold change from the control (time 0 hr) of the MDI + DMSO group. (c) The scheme of probe selection for further clustering. Genes that had a fold change ≥ 2.0 (x ≥ 1) at both 2 hr and 6 hr relative to time 0 preadipocytes were further analysed using hierarchical clustering. ( C ) Representative quantitative RT-PCR analysis of Cebpb , Cebpd , Klf4 , and Klf5 were performed to validate the microarray analysis. The data are the mean ± SEM of three independent experiments. *P

    Journal: Scientific Reports

    Article Title: Echinomycin inhibits adipogenesis in 3T3-L1 cells in a HIF-independent manner

    doi: 10.1038/s41598-017-06761-4

    Figure Lengend Snippet: Time-course DNA microarray analysis of 3T3-L1 cells treated with MDI plus DMSO or MDI plus echinomycin. ( A ) Clustering analysis performed among the MDI + DMSO treated groups using 2,204 selected probes that had a fold change ≥ 2.0 in expression compared with the control (0 hr) (See the details in Methods). Genes were clustered into 3 groups according to their timing of response to MDI treatment. ( B ) (a,b) Scatter plot of all the 14,005 probes (signal intensity ≥ 100 at any one point) at 2 hr and 6 hr. A log2-fold change from the control (time 0 hr) of the MDI + echinomycin group is plotted against a log2-fold change from the control (time 0 hr) of the MDI + DMSO group. (c) The scheme of probe selection for further clustering. Genes that had a fold change ≥ 2.0 (x ≥ 1) at both 2 hr and 6 hr relative to time 0 preadipocytes were further analysed using hierarchical clustering. ( C ) Representative quantitative RT-PCR analysis of Cebpb , Cebpd , Klf4 , and Klf5 were performed to validate the microarray analysis. The data are the mean ± SEM of three independent experiments. *P

    Article Snippet: Cebpb promoter fragments of 3 kb, 2 kb, 1 kb, or 0.5 kb (mCEBPB3K, 2 K, 1 K, or 0.5 K promoter) were PCR amplified from mouse genomic DNA using PrimeStar HS DNA polymerase (Takara Bio, Shiga, Japan) and primers with KpnI and XhoI overhangs (see Table for primers).

    Techniques: Microarray, Expressing, Selection, Quantitative RT-PCR

    The effect of PPARδ activation on ischemia-induced cerebrovascular permeability and potential mechanisms in vivo . Mice were pretreated with a specific PPARδ agonist, GW 501516, or vehicle reagent for 24h via mini-pump assisted-cerebroventricular infusion, and then subjected to 1h MCA occlusion and 24h-reperfusion. 2% TTC-stained coronal sections were shown at different brain levels posterior to the frontal pole (A). Quantitative analysis was made on brain infarct volume (B), and cerebrovascular permeability was determined 1h after injection from Evans Blue extravasation as described in the Methods (C) (n=6) in mice after stroke. In comparison to the vehicle control, treatment of GW 501516 significantly attenuates ischemic brain infarction and improves cerebrovascular permeability. ICV infusion of GW 501516 also significantly reduced ischemia-induced miR-15a upregulation (D), increased bcl-2 protein levels (E), and attenuated caspase-3 activation (F) and DNA fragmentation (G) in the cerebral microvasculature in mice after MCAO. Data are expressed as mean ± SD. * p

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: PPAR? regulation of miR-15a in ischemia-induced cerebral vascular endothelial injury

    doi: 10.1523/JNEUROSCI.0780-10.2010

    Figure Lengend Snippet: The effect of PPARδ activation on ischemia-induced cerebrovascular permeability and potential mechanisms in vivo . Mice were pretreated with a specific PPARδ agonist, GW 501516, or vehicle reagent for 24h via mini-pump assisted-cerebroventricular infusion, and then subjected to 1h MCA occlusion and 24h-reperfusion. 2% TTC-stained coronal sections were shown at different brain levels posterior to the frontal pole (A). Quantitative analysis was made on brain infarct volume (B), and cerebrovascular permeability was determined 1h after injection from Evans Blue extravasation as described in the Methods (C) (n=6) in mice after stroke. In comparison to the vehicle control, treatment of GW 501516 significantly attenuates ischemic brain infarction and improves cerebrovascular permeability. ICV infusion of GW 501516 also significantly reduced ischemia-induced miR-15a upregulation (D), increased bcl-2 protein levels (E), and attenuated caspase-3 activation (F) and DNA fragmentation (G) in the cerebral microvasculature in mice after MCAO. Data are expressed as mean ± SD. * p

    Article Snippet: A 1,887 bp segment from the promoter region (−1824/+62) of the mouse miR-15a gene (miR-15a 1.9 kb Wt) was amplified by PCR from mouse genomic DNA and then cloned into the Kpn I/Xho I site of the pGL 4.10 Luciferase vector (Promega).

    Techniques: Activation Assay, Permeability, In Vivo, Mouse Assay, Staining, Injection

    Functional analysis of a PPRE site located in the mouse miR-15a promoter. (A) Schematic representation of the mouse miR-15a promoter region. A putative PPARδ binding site was demonstrated in the promoter of miR-15a at locations of −1593/−1573 bp (PPRE). The translation start site is numbered as +1. (B) Luciferase reporter assays were performed by transfecting HEK 293 cells with a miR-15a 1.9 kb wild-type promoter (miR-15a 1.9 kb Wt) versus a PPRE mutated miR-15a promoter (miR-15a 1.9 kb PPRE mut.). Transcriptional activity is reduced upon overexpression of PPARδ in the miR-15a wild-type but not in the PPRE mutant promoter. (C) A ChIP assay showing PPARδ binds to the putative PPRE site in the mouse miR-15a promoter. Mouse CECs were infected with an adenovirus carrying GFP or PPARδ for 48h. DNA from PPARδ immunoprecipitated chromatin and input were subjected to PCR analysis using a pair of primer covering regions containing the PPRE site in the mouse miR-15a promoter. The experiments were repeated at least three times. Data were expressed as mean ± SD. * P

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: PPAR? regulation of miR-15a in ischemia-induced cerebral vascular endothelial injury

    doi: 10.1523/JNEUROSCI.0780-10.2010

    Figure Lengend Snippet: Functional analysis of a PPRE site located in the mouse miR-15a promoter. (A) Schematic representation of the mouse miR-15a promoter region. A putative PPARδ binding site was demonstrated in the promoter of miR-15a at locations of −1593/−1573 bp (PPRE). The translation start site is numbered as +1. (B) Luciferase reporter assays were performed by transfecting HEK 293 cells with a miR-15a 1.9 kb wild-type promoter (miR-15a 1.9 kb Wt) versus a PPRE mutated miR-15a promoter (miR-15a 1.9 kb PPRE mut.). Transcriptional activity is reduced upon overexpression of PPARδ in the miR-15a wild-type but not in the PPRE mutant promoter. (C) A ChIP assay showing PPARδ binds to the putative PPRE site in the mouse miR-15a promoter. Mouse CECs were infected with an adenovirus carrying GFP or PPARδ for 48h. DNA from PPARδ immunoprecipitated chromatin and input were subjected to PCR analysis using a pair of primer covering regions containing the PPRE site in the mouse miR-15a promoter. The experiments were repeated at least three times. Data were expressed as mean ± SD. * P

    Article Snippet: A 1,887 bp segment from the promoter region (−1824/+62) of the mouse miR-15a gene (miR-15a 1.9 kb Wt) was amplified by PCR from mouse genomic DNA and then cloned into the Kpn I/Xho I site of the pGL 4.10 Luciferase vector (Promega).

    Techniques: Functional Assay, Binding Assay, Luciferase, Activity Assay, Over Expression, Mutagenesis, Chromatin Immunoprecipitation, Infection, Immunoprecipitation, Polymerase Chain Reaction

    Alignment of JSRV JS7 provirus restriction maps. (A) Results of Southern blot analysis of JS7 genomic DNA digested with restriction endonucleases Eco RI (E), Hin dIII (H), and Sac ). (B) Full-length proviral lambda clone 2-1. (C) Partial-length proviral lambda clone 5-1.

    Journal: Journal of Virology

    Article Title: Jaagsiekte Sheep Retrovirus Proviral Clone JSRVJS7, Derived from the JS7 Lung Tumor Cell Line, Induces Ovine Pulmonary Carcinoma and Is Integrated into the Surfactant Protein A Gene

    doi: 10.1128/JVI.75.9.4239-4246.2001

    Figure Lengend Snippet: Alignment of JSRV JS7 provirus restriction maps. (A) Results of Southern blot analysis of JS7 genomic DNA digested with restriction endonucleases Eco RI (E), Hin dIII (H), and Sac ). (B) Full-length proviral lambda clone 2-1. (C) Partial-length proviral lambda clone 5-1.

    Article Snippet: Genomic DNA (100 μg) from JS7 cells (passage 170) was partially digested with 0.85 × 10−3 U of restriction endonuclease Sau 3A per μg for 30 min at 37°C to generate fragments between 12 and 23 kb; these were ligated to Bam HI-digested arms of the lambda BlueStar vector (Novagen).

    Techniques: Southern Blot