la pcr genome dna set  (TaKaRa)

Journal: Molecular and Cellular Biology

Article Title: HuR Maintains a Replicative Life Span by Repressing the ARF Tumor Suppressor

doi: 10.1128/MCB.01277-12

Figure Lengend Snippet: Adipose-specific HuR deletion accelerates the senescence of adipocytes. (A) Genotyping of adipose- and tail-derived genomic DNA. (B) HuR mRNA levels in adipose tissue from mice with the indicated genotypes were analyzed by real-time PCR. Values were normalized

Article Snippet: RNAs were prepared from cells or immune complexes using TriPure isolation reagent (Roche, Indianapolis, IN), reverse transcribed using a PrimeScript reverse transcriptase (RT) reagent kit with the genomic DNA (gDNA) Eraser (TaKaRa, Shiga, Japan), and subjected to PCR using the following primers: for PAI-1 , 5′-TCAGAGCAACAAGTTCAACTACACTGAG-3′ (sense) and 5′-CCCACTGTCAAGGCTCCATCACTTGCCCCA-3′ (antisense); for HuR , 5′-TTGGGCTACGGTTTTGTGAAC-3′ (sense) and 5′-CCCACTGATGTATAAGTTGGCAT-3′ (antisense); for ARF , 5′-GCCGCACCGGAATCCT-3′ (sense) and 5′-TTGAGCAGAAGAGCTGCTACGT-3′ (antisense); for Ink4a , 5′-CCCAACGCCCCGAACT-3′ (sense) and 5′-GCAGAAGAGCTGCTACGTGAA-3′ (antisense); for c- myc , 5′-TCTATTTGGGGACAGTGTTC-3′ (sense) and 5′-GGTCATAGTTCCTGTTGGTG-3′ (antisense); for p53 , 5′-TGGAGAGTATTTCACCCTCAAGA-3′ (sense) and 5′-CTCCTCTGTAGCATGGGCATC-3′ (antisense); for β-actin , 5′-CTAAGGCCAACCGTGAAAAG-3′ (sense) and 5′-ACCAGAGGCATACAGGGACA-3′ (antisense); for 18S rRNA 5′-AGTCCCTGCCCTTTGTACACA-3′ (sense) and 5′-GATCCGAGGGCCTCACTAAAC-3′ (antisense); for AUF-1 , 5′-TTTCTCCAGACACACCTGAAGA-3′ (sense) and 5′-CTGTTCCTTTGACATGGCTACTT-3′ (antisense); and for GFP , 5′-TCTGCACCACCGGCAAGCTG-3′ (sense) and 5′-TGCGCTCCTGGACGTAGCCT-3′ (antisense).

Techniques: Derivative Assay, Mouse Assay, Real-time Polymerase Chain Reaction

Journal: Molecular Genetics & Genomic Medicine

Article Title: Spinal muscular atrophy caused by a novel Alu‐mediated deletion of exons 2a‐5 in SMN1 undetectable with routine genetic testing, et al. Spinal muscular atrophy caused by a novel Alu‐mediated deletion of exons 2a‐5 in SMN1 undetectable with routine genetic testing

doi: 10.1002/mgg3.1238

Figure Lengend Snippet: Identification of SMN1 variants. (a) qPCR analysis of four retrotransposon‐free SMN genomic regions in the intron 1 (I1), in the exon 3–intron 3 junction (E3I3), in the intron 5–exon 6 junction (I5E6), and ~1 kb downstream from exon 8 (+1 kb). Compared with four copies of SMN genes that are present in controls, we found that the patient has three copies at the I1, I5E6, and +1 kb loci and two copies at the E3I3 loci. The mother has three copies through the I1 to I5E6 loci and two copies at the +1 kb loci. The father has three copies only at the I5E6 loci. (b) Schematic representation of SMN1/2 exon (E)/intron (I) structure. Positions of sequence differences between SMN1 and SMN2 are represented by black vertical bars. The black triangles denote sequence‐specific variants in exons 7, 8 targeted by MLPA probes in routine testing. Locations of Alus in the breakpoint candidate regions in the intron 1 and 5, including the causal AluSp in the intron 1 and AluSq in the intron 5 indicated by vertical text, and primers binding sites for Alu PCR indicated by black arrowheads are shown below the scheme of the SMN structure. Position of the PCR4 spanning exons 5–8 that showed absence of SMN1 sequence‐specific variants indicating disruption of both SMN1 alleles in the patient is represented by yellow box. Range of the paternal deletion of exons 2a‐5 is represented by red box. (c) DNA sequence trace of the Alu PCR, Alu_259_4A, showing a double sequence caused by presence of AluSq wt in intron 5 together with a sequence originating from the intron 1 AluSp . Red arrows indicate the addition of AluSp ‐specific sequence in an Alu PCR product. (d) PCR genotyping of the SMN1Δ(2a‐5) variant showed presence of the deletion‐spanning amplification product in the patient (P) and father (F), but not in mother (M) and control (C). (e) DNA sequence trace of the breakpoint junction‐specific PCR and detail of the Δ2a‐5 breakpoint junction show the new Alu ‐ Alu chimeric element originating from the recombination between the AluSp in the intron 1 and AluSq in the intron 5. A breakpoint microhomology of the AluSp and AluSq is marked with a black box. (f) Schematic representation of SMN1 and SMN2 in the family members. Pink‐marked boxes represent maternal alleles (M) and blue boxes paternal alleles (F). The red crosses denote identified deletions and the dashed vertical lines denote loci of the qPCR (I1, E3I3, I5E6, and +1 kb) and MLPA (exon 7‐E7, exon 8‐E8) probes used for deletion mapping. The black junctions on the box terminals indicate a cis configuration of SMN1 and SMN2 alleles. The model shows (a) a whole deletion of one SMN1 allele in the patient (P) inherited from her mother and detected by the combination of the qPCR and MLPA; (b) a deletion of the second SMN1 allele in the patient inherited from her father and detected by the E3I3 qPCR and transcript analysis (Figure 1a ); and (c) deletion of one copy of one SMN2 allele in the mother detected by the MLPA and the + 1kb qPCR

Article Snippet: 2.6 Long‐range PCR Long‐range PCR was performed using four primer pairs amplifying both SMN genes (NG_008691.1, NG_008728.1) in four overlapping PCR products (PCR1‐PCR4, Table ).

Techniques: Real-time Polymerase Chain Reaction, Sequencing, Multiplex Ligation-dependent Probe Amplification, Binding Assay, Polymerase Chain Reaction, Variant Assay, Amplification

Journal: PLoS ONE

Article Title: Non-invasive in vivo imaging of UCP1 expression in live mice via near-infrared fluorescent protein iRFP720

doi: 10.1371/journal.pone.0225213

Figure Lengend Snippet: Generation of Ucp1-iRFP720 KI mice. (A) Targeting of the iRFP720 gene into the Ucp1 locus. The donor vector contains an artificial intron (orange box), iRFP720 gene (red box) and the bovine growth factor gene-derived polyadenylation signal (BGHpA) (green box). Structure of the targeted allele is indicated together with the primers used for genomic PCR (red arrows). (B) Genomic PCR analysis of F0 mice carrying the Ucp1-iRFP720 KI allele. M, marker; wt, wild-type; ctrl, positive control (a DNA fragment that covers the amplicon sequence between the primers c and f in Fig 2A). (C) PCR analysis of the donor vector and the gRNA/Cas9-expression vector randomly integrated into the F0 mouse genome. The table shows the summary of random integration in the F0 mouse genome carrying the Ucp1-iRFP720 allele. (D) Mendelian inheritance of the Ucp1-iRFP720 KI allele. One of the Ucp1-iRFP720 KI mice (#71) was crossed with a WT mouse, and eight F1 mice were born. Their genotypes were analyzed by PCR using the primer sets shown in Fig 2A. Small white and red rectangles show WT Ucp1 and Ucp1-iRFP720 KI alleles, respectively. Rounded rectangles and ellipses indicate male and female mice, respectively.

Article Snippet: To confirm the correct insertion of the iRFP720 gene at the Ucp1 locus, 10 ng each of the genome DNA was analyzed by PCR using 0.25 unit of PrimeStar GXL DNA Polymerase (TaKaRa Bio Inc.) and 3 pmoles each of the primers in a 10 μl reaction.

Techniques: Mouse Assay, Plasmid Preparation, Derivative Assay, Polymerase Chain Reaction, Marker, Positive Control, Amplification, Sequencing, Expressing

Journal: PLoS ONE

Article Title: Non-invasive in vivo imaging of UCP1 expression in live mice via near-infrared fluorescent protein iRFP720

doi: 10.1371/journal.pone.0225213

Figure Lengend Snippet: Selection of the optimum gRNA sequence to generate Ucp1-iRFP720 KI mice. (A) Structure of the mouse Ucp1 gene. The boxes and the numbers above each box indicate the exons of the mouse Ucp1 gene. Light and dark gray regions within the exons indicate untranslated and translated regions, respectively. The position of the transcription start site is indicated by +1. The DNA sequence around the translation start site of the mouse Ucp1 gene is shown. The red arrows indicate the target sequences of the gRNAs (dashed arrows indicate the complementary sequences). The red boxes show the protospacer adjacent motif (PAM) sequences. (B) The assay system for estimating the functional genome editing efficiency of gRNAs. The Ucp1 gene (+1 ~ +500) containing the gRNA target sites (shown in orange) was inserted into pCAG-EGxxFP vector. When recruited by the gRNA (shown in dark blue) to the target site, the Cas9 nuclease (shown in light blue) introduces a double strand break in the target DNA. Because of homologous recombination between the duplicated sequences (shown in dark green), the EGFP-coding gene is recovered. (C) Fluorescence image of HEK293T cells two days after transfection. The Cas9 nuclease was expressed together with one of the four gRNA candidates (gRNA1~gRNA4) or without gRNA (Cas9 only). Scale bar, 200 μm.

Article Snippet: To confirm the correct insertion of the iRFP720 gene at the Ucp1 locus, 10 ng each of the genome DNA was analyzed by PCR using 0.25 unit of PrimeStar GXL DNA Polymerase (TaKaRa Bio Inc.) and 3 pmoles each of the primers in a 10 μl reaction.

Techniques: Selection, Sequencing, Mouse Assay, Functional Assay, Plasmid Preparation, Homologous Recombination, Fluorescence, Transfection

Journal: Molecular Therapy. Nucleic Acids

Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP

doi: 10.1016/j.omtn.2018.04.008

Figure Lengend Snippet: Sp1 Negatively Regulates miR-3178 by Binding to Its Promoter Region (A and B) miRNA profiling analysis. LNCaP cells were treated with 100 nM bortezomib (BTZ) (A) or 2.5 μM celastrol (CEL) (B) for 12 hr. miRNAs with over 2-fold changes against control were shown. (C) Expressions of SP1 and miR-3178 post-treatments as (A) and (B) in LNCaP cells are shown. (D) Consensus Sp1 sites and predicted binding sites (BSs) of Sp1 in miR-3178 promoter are shown. Mutations of each BS were indicated by italic red cases. (E) Luciferase reporter assay is shown. Luciferase reporter constructs were generated as schematic depiction and transfected into 1E8 cells in the presence of SP1 or control (Ctrl) plasmid. Wild-type (WT) and mutant Sp1 sites (Mut1–3) were indicated by blank and italic dash, respectively. NS, non-significant difference. (F) ChIP assay is shown. DNA was immunoprecipitated with anti-IgG or anti-Sp1 antibody and amplified by PCR using primer specific for BS3. Input chromatin before immunoprecipitation was used as control. Experiments were repeated three times, and results were shown as mean ± SD in the lower panel. (G) Expressions of miR-3178 in prostate, lung, and breast cancer cell lines are shown. (H) Expression of miR-3178 in lowly metastatic cancer cells after ectopic expression of SP1 is shown. Values were shown as mean ± SD of three independent experiments. *p

Article Snippet: Plasmid Construction and Cell Transfection DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan).

Techniques: Binding Assay, Luciferase, Reporter Assay, Construct, Generated, Transfection, Plasmid Preparation, Mutagenesis, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Expressing