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  • 99
    Thermo Fisher chargeswitch pcr clean up kit
    Chargeswitch Pcr Clean Up Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 469 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore genelute pcr clean up kit
    Genelute Pcr Clean Up Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up system
    Screening and generation of Mp erf13 mutants (A) The flow chart of the screen for mutants defective in oil body formation from <t>T-DNA-insertion</t> lines. (B) A maximum likelihood phylogenetic tree of proteins with one ERF/AP2 domain. The color code is shown in Fig S1A . The branch lengths are proportional to the estimated number of substitutions per site. Bootstrap probability is indicated as a percentage on each branch with at least 50% support. A more detailed tree was presented previously ( 20 ). (C) Schematic representation of the Mp ERF13 gene structure and mutations generated in this study. Gray and black boxes indicate the UTR and coding sequences, respectively. Asterisks with numbers indicate sites of designed gRNA to generate Mp erf13-1 ge (*1 and *4) and Mp erf13-2 ge (*2 and *3). (D) <t>PCR-based</t> genotyping of Tak-1, Mp erf13 GOF , Mp erf13-1 ge , and Mp erf13-2 ge . The combinations and annealing sites of primers (a to d) are shown in (C). (E) The genomic and predicted amino acid sequences of the Mp ERF13 locus in Tak-1 and Mp erf13 mutants. Light blue, dark blue, and magenta letters indicate UTR, coding, and predicted amino acid sequences, respectively. The PAM sequences for gRNAs are underlined. The caret indicates the indel site. (F) Fluorescent and bright-field images of BODIPY-stained three-week-old thalli of Tak-1, Mp erf13-1 ge , and Mp erf13-2 ge . Bars = 0.5 mm. (G) The number of oil bodies visualized with BODIPY in a unit area (2.0 mm × 2.0 mm). Bars indicate means ± SD. Statistical analyses between Tak-1 and each genotype were conducted using a two-tailed Welch’s t -test. Sample numbers were 23 thalli for Tak-1, 24 for Mp erf13-1 ge , and 26 for Mp erf13-2 ge . p -values are 5.14×10 −11 for Mp erf13-1 ge and 5.14×10 −11 for Mp erf13-2 ge .
    Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 22798 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL pcr clean up kit
    Host and viral <t>DNA</t> content of differently treated ASFV samples. Quantitative dual <t>PCR</t> was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.
    Pcr Clean Up Kit, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 94/100, based on 6126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega wizard sv gel
    Illustrative representation of HSP 70 (234) amplification for Leishmania sp. before and after re-amplification of the <t>PCR</t> products. (A) PCR products of first amplification of HSP 70 (234) targeting analyzed by electrophoresis polyacrylamide gel stained with silver. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Infected Thrichomys laurentius from São Raimundo Nonato/PI; 2. Negative control of PCR reaction. (B) PCR products of re-amplification of the product obtained in the first PCR reaction. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Negative Thrichomys fosteri from Corumbá/MS (Positive only in kDNA); 2–5. Infected T. laurentius from São Raimundo Nonato/PI; 6. Infected T. fosteri from Corumbá/MS; 7. Negative control of PCR after re-amplification.
    Wizard Sv Gel, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 4009 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL pcr clean up
    Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific <t>PCR</t> analysis of the <t>chd</t> gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.
    Pcr Clean Up, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 94/100, based on 1731 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen ultraclean pcr clean up kit
    Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific <t>PCR</t> analysis of the <t>chd</t> gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.
    Ultraclean Pcr Clean Up Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1693 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen pcr clean up kit
    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic <t>DNA</t> purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using <t>PCR.</t> b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses
    Pcr Clean Up Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1848 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up kit
    Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by <t>PCR</t> using primers specific to these regions (a). L-100 bp ladder. PCR products were sequenced by Sangers method. All the 4 nucleotide differences were also present at genomic <t>DNA</t> level. Representative image of exon specific amplification (b). Alignment of exon specific sequencing results with human, mouse, and rat (predicted) sequence shows 2 amino acid residue changes (c).
    Pcr Clean Up Kit, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 1399 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa nucleospin gel
    Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the <t>NucleoSpin</t> Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.
    Nucleospin Gel, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pcr clean up kit
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain <t>DNA</t> (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Pcr Clean Up Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 830 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axygen axyprep mag pcr clean up
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain <t>DNA</t> (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Axyprep Mag Pcr Clean Up, supplied by Axygen, used in various techniques. Bioz Stars score: 90/100, based on 640 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axygen axyprep mag pcr clean up kit
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain <t>DNA</t> (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Axyprep Mag Pcr Clean Up Kit, supplied by Axygen, used in various techniques. Bioz Stars score: 89/100, based on 474 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Axygen axyprep mag pcr clean up beads
    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain <t>DNA</t> (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time <t>RT-PCR</t> of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition
    Axyprep Mag Pcr Clean Up Beads, supplied by Axygen, used in various techniques. Bioz Stars score: 90/100, based on 209 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MACHEREY NAGEL pcr clean up columns
    Characterization of a stable <t>eys</t> rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of <t>RT-PCR</t> analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.
    Pcr Clean Up Columns, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 89/100, based on 240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega wizard sv pcr clean up system
    Diagrammatic representation of the RNA silencing constructs (not drawn to scale). (A) A 21 nucleotide artificial microRNA (ami-BEIIb)-based osa -miR528 was synthesized by fusion <t>PCR</t> and cloned into Vec8, a Ti binary vector with a wheat high molecular weight glutenin promoter (wHMGPro) and nopaline synthase terminator (NOS). (B) The secondary structure of the osa -miR528 backbone as predicted by RNAfold, including information on ami-BEIIb (reverse complement). The predicted target cleavage site (arrow with sequence bold and highlighted) is located between positions 10 and 11 of the <t>amiRNA,</t> while the two mismatches (grey highlight) are located at positions 1 and 21. (C) The hairpin RNA (hp-BEIIb) was cloned in Vec8 by inserting a 397 bp BEIIb fragment in the sense (BEIIb→) and antisense (BEIIb←) orientations. The two fragments are flanked by two rice introns, Rint4 and Rint9, which form a hairpin loop. The amiRNA and hp-RNA fragments were directionally cloned using several restriction sites (H, Hin dIII; B, Bam HI; K, Kpn I; N, Not I; E Eco RI; S, Spe I).
    Wizard Sv Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Screening and generation of Mp erf13 mutants (A) The flow chart of the screen for mutants defective in oil body formation from T-DNA-insertion lines. (B) A maximum likelihood phylogenetic tree of proteins with one ERF/AP2 domain. The color code is shown in Fig S1A . The branch lengths are proportional to the estimated number of substitutions per site. Bootstrap probability is indicated as a percentage on each branch with at least 50% support. A more detailed tree was presented previously ( 20 ). (C) Schematic representation of the Mp ERF13 gene structure and mutations generated in this study. Gray and black boxes indicate the UTR and coding sequences, respectively. Asterisks with numbers indicate sites of designed gRNA to generate Mp erf13-1 ge (*1 and *4) and Mp erf13-2 ge (*2 and *3). (D) PCR-based genotyping of Tak-1, Mp erf13 GOF , Mp erf13-1 ge , and Mp erf13-2 ge . The combinations and annealing sites of primers (a to d) are shown in (C). (E) The genomic and predicted amino acid sequences of the Mp ERF13 locus in Tak-1 and Mp erf13 mutants. Light blue, dark blue, and magenta letters indicate UTR, coding, and predicted amino acid sequences, respectively. The PAM sequences for gRNAs are underlined. The caret indicates the indel site. (F) Fluorescent and bright-field images of BODIPY-stained three-week-old thalli of Tak-1, Mp erf13-1 ge , and Mp erf13-2 ge . Bars = 0.5 mm. (G) The number of oil bodies visualized with BODIPY in a unit area (2.0 mm × 2.0 mm). Bars indicate means ± SD. Statistical analyses between Tak-1 and each genotype were conducted using a two-tailed Welch’s t -test. Sample numbers were 23 thalli for Tak-1, 24 for Mp erf13-1 ge , and 26 for Mp erf13-2 ge . p -values are 5.14×10 −11 for Mp erf13-1 ge and 5.14×10 −11 for Mp erf13-2 ge .

    Journal: bioRxiv

    Article Title: Switching secretory pathway direction for organelle acquisition in plants

    doi: 10.1101/2020.03.02.956961

    Figure Lengend Snippet: Screening and generation of Mp erf13 mutants (A) The flow chart of the screen for mutants defective in oil body formation from T-DNA-insertion lines. (B) A maximum likelihood phylogenetic tree of proteins with one ERF/AP2 domain. The color code is shown in Fig S1A . The branch lengths are proportional to the estimated number of substitutions per site. Bootstrap probability is indicated as a percentage on each branch with at least 50% support. A more detailed tree was presented previously ( 20 ). (C) Schematic representation of the Mp ERF13 gene structure and mutations generated in this study. Gray and black boxes indicate the UTR and coding sequences, respectively. Asterisks with numbers indicate sites of designed gRNA to generate Mp erf13-1 ge (*1 and *4) and Mp erf13-2 ge (*2 and *3). (D) PCR-based genotyping of Tak-1, Mp erf13 GOF , Mp erf13-1 ge , and Mp erf13-2 ge . The combinations and annealing sites of primers (a to d) are shown in (C). (E) The genomic and predicted amino acid sequences of the Mp ERF13 locus in Tak-1 and Mp erf13 mutants. Light blue, dark blue, and magenta letters indicate UTR, coding, and predicted amino acid sequences, respectively. The PAM sequences for gRNAs are underlined. The caret indicates the indel site. (F) Fluorescent and bright-field images of BODIPY-stained three-week-old thalli of Tak-1, Mp erf13-1 ge , and Mp erf13-2 ge . Bars = 0.5 mm. (G) The number of oil bodies visualized with BODIPY in a unit area (2.0 mm × 2.0 mm). Bars indicate means ± SD. Statistical analyses between Tak-1 and each genotype were conducted using a two-tailed Welch’s t -test. Sample numbers were 23 thalli for Tak-1, 24 for Mp erf13-1 ge , and 26 for Mp erf13-2 ge . p -values are 5.14×10 −11 for Mp erf13-1 ge and 5.14×10 −11 for Mp erf13-2 ge .

    Article Snippet: After agarose gel electrophoresis of the final TAIL-PCR products, DNA bands were excised and purified using the Wizard SV Gel and PCR Clean-Up System (Promega).

    Techniques: Generated, Polymerase Chain Reaction, Staining, Two Tailed Test

    Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.

    Journal: Viruses

    Article Title: A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus

    doi: 10.3390/v11121129

    Figure Lengend Snippet: Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.

    Article Snippet: Amplified DNA Clean Up REPLI-g samples were purified using the NucleoSpin Gel and PCR clean-up Kit (Macherey-Nagel Düren, Germany).

    Techniques: Polymerase Chain Reaction, Whole Genome Amplification

    Illustrative representation of HSP 70 (234) amplification for Leishmania sp. before and after re-amplification of the PCR products. (A) PCR products of first amplification of HSP 70 (234) targeting analyzed by electrophoresis polyacrylamide gel stained with silver. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Infected Thrichomys laurentius from São Raimundo Nonato/PI; 2. Negative control of PCR reaction. (B) PCR products of re-amplification of the product obtained in the first PCR reaction. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Negative Thrichomys fosteri from Corumbá/MS (Positive only in kDNA); 2–5. Infected T. laurentius from São Raimundo Nonato/PI; 6. Infected T. fosteri from Corumbá/MS; 7. Negative control of PCR after re-amplification.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Distinct Leishmania Species Infecting Wild Caviomorph Rodents (Rodentia: Hystricognathi) from Brazil

    doi: 10.1371/journal.pntd.0003389

    Figure Lengend Snippet: Illustrative representation of HSP 70 (234) amplification for Leishmania sp. before and after re-amplification of the PCR products. (A) PCR products of first amplification of HSP 70 (234) targeting analyzed by electrophoresis polyacrylamide gel stained with silver. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Infected Thrichomys laurentius from São Raimundo Nonato/PI; 2. Negative control of PCR reaction. (B) PCR products of re-amplification of the product obtained in the first PCR reaction. Lanes: bp. molecular-weight marker (50 bp DNA ladder); 1. Negative Thrichomys fosteri from Corumbá/MS (Positive only in kDNA); 2–5. Infected T. laurentius from São Raimundo Nonato/PI; 6. Infected T. fosteri from Corumbá/MS; 7. Negative control of PCR after re-amplification.

    Article Snippet: The PCR products obtained for HSP70 (234) targeting and the products positive only in kDNA targeting were purified using the Wizard SV Gel kit and PCR Clean-up System kit (Promega).

    Techniques: Amplification, Polymerase Chain Reaction, Electrophoresis, Staining, Molecular Weight, Marker, Infection, Negative Control, Mass Spectrometry

    High-throughput sequencing and mapping to α-satellite DNA reveals that centromeric CENP-A chromatin is an octameric nucleosome with transient DNA unwrapping of the DNA entry and exit sites at G1, G2, and mitosis. (A) Experimental design for obtaining CENP-A TAP – and H3.1 TAP -bound DNA sequences. (B) Microcapillary electrophoresis of MNase-digested bulk input mononucleosomes (top) and CENP-A TAP or H3.1 TAP or NH2 H3 CATD native ChIP (middle and bottom, immunoprecipitation). Purified CENP-A chromatin is nucleosome-like but protects a shorter DNA length than does H3.1-containing nucleosomes. (C) Quantitative real-time PCR for α-satellite DNA extracted from CENP-A TAP chromatin in random cycling (RC; magenta), G1 (red), and G2 (blue). n = 2 from two independent replicates. Error bars represent SEM. (D) CENP-A TAP – and H3.1 TAP -bound DNA was sequenced using paired-end 100-bp ChIP sequencing and then mapped to the human genome 38 assembly (hg38), which contains α-satellite sequence models for each centromere. Approximately 50% of CENP-A TAP –bound DNA is centromeric at G1 and G2. (E) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (F) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA on chromosome X were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (G) Distribution of the chromatin DNA lengths into two length bins: subnucleosomal (60–85 bp, as predicted for tetrasomes and hemisomes; Hasson et al., 2013 ) and nucleosomal (100–170 bp) for bulk input chromatin and affinity-purified CENP-A TAP and H3.1 TAP chromatin mapped to α-satellite DNA. n = 2 from two independent replicates. Error bars represent SEM.

    Journal: The Journal of Cell Biology

    Article Title: Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

    doi: 10.1083/jcb.201608083

    Figure Lengend Snippet: High-throughput sequencing and mapping to α-satellite DNA reveals that centromeric CENP-A chromatin is an octameric nucleosome with transient DNA unwrapping of the DNA entry and exit sites at G1, G2, and mitosis. (A) Experimental design for obtaining CENP-A TAP – and H3.1 TAP -bound DNA sequences. (B) Microcapillary electrophoresis of MNase-digested bulk input mononucleosomes (top) and CENP-A TAP or H3.1 TAP or NH2 H3 CATD native ChIP (middle and bottom, immunoprecipitation). Purified CENP-A chromatin is nucleosome-like but protects a shorter DNA length than does H3.1-containing nucleosomes. (C) Quantitative real-time PCR for α-satellite DNA extracted from CENP-A TAP chromatin in random cycling (RC; magenta), G1 (red), and G2 (blue). n = 2 from two independent replicates. Error bars represent SEM. (D) CENP-A TAP – and H3.1 TAP -bound DNA was sequenced using paired-end 100-bp ChIP sequencing and then mapped to the human genome 38 assembly (hg38), which contains α-satellite sequence models for each centromere. Approximately 50% of CENP-A TAP –bound DNA is centromeric at G1 and G2. (E) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (F) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA on chromosome X were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (G) Distribution of the chromatin DNA lengths into two length bins: subnucleosomal (60–85 bp, as predicted for tetrasomes and hemisomes; Hasson et al., 2013 ) and nucleosomal (100–170 bp) for bulk input chromatin and affinity-purified CENP-A TAP and H3.1 TAP chromatin mapped to α-satellite DNA. n = 2 from two independent replicates. Error bars represent SEM.

    Article Snippet: DNA was purified from proteinase K–treated samples using a DNA purification kit following the manufacturer instructions (A9282; Promega) and was subsequently analyzed either by running a 2% low melting agarose (APEX) gel or by an Agilent Technologies 2100 Bioanalyzer by using the DNA 1000 kit.

    Techniques: Next-Generation Sequencing, Electrophoresis, Chromatin Immunoprecipitation, Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction, Sequencing, Affinity Purification

    CENP-A chromatin has the physical characteristics of nucleosomes. (A) Experimental design for the separation of in vitro–reconstituted octameric nucleosomes or tetrasomes over a 5–25% sucrose gradient. (B) After sedimentation of in vitro–reconstituted H3 or CENP-A octameric nucleosomes or (CENP-A/H4) 2 or (H3/H4) 2 tetrasomes, fractions were immunoblotted for CENP-A or H3. (C) Experimental design for the sedimentation of in vivo bulk nucleosomes and short polynucleosomes at different points of the cell cycle. (D) Moderate MNase digestion profile of bulk chromatin from random cycling (RC) cells expressing CENP-A TAP or H3.1 TAP . (E) Sucrose gradient sedimentation and fractionation of bulk nucleosomes from CENP-A TAP –expressing cells synchronized at G1, G2, and mitosis. (top) Ethidium bromide stained DNA agarose gel revealing the DNA length extracted from the different fractions. (bottom) Immunoblot for CENP-A TAP . (F) Real-time quantitative PCR for α-satellite DNA in the different fractions (colored). Second axis shows quantification of CENP-A immunoblot shown in E. No CENP-A or α-satellite DNA was detected in fractions 7 and 9.

    Journal: The Journal of Cell Biology

    Article Title: Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

    doi: 10.1083/jcb.201608083

    Figure Lengend Snippet: CENP-A chromatin has the physical characteristics of nucleosomes. (A) Experimental design for the separation of in vitro–reconstituted octameric nucleosomes or tetrasomes over a 5–25% sucrose gradient. (B) After sedimentation of in vitro–reconstituted H3 or CENP-A octameric nucleosomes or (CENP-A/H4) 2 or (H3/H4) 2 tetrasomes, fractions were immunoblotted for CENP-A or H3. (C) Experimental design for the sedimentation of in vivo bulk nucleosomes and short polynucleosomes at different points of the cell cycle. (D) Moderate MNase digestion profile of bulk chromatin from random cycling (RC) cells expressing CENP-A TAP or H3.1 TAP . (E) Sucrose gradient sedimentation and fractionation of bulk nucleosomes from CENP-A TAP –expressing cells synchronized at G1, G2, and mitosis. (top) Ethidium bromide stained DNA agarose gel revealing the DNA length extracted from the different fractions. (bottom) Immunoblot for CENP-A TAP . (F) Real-time quantitative PCR for α-satellite DNA in the different fractions (colored). Second axis shows quantification of CENP-A immunoblot shown in E. No CENP-A or α-satellite DNA was detected in fractions 7 and 9.

    Article Snippet: DNA was purified from proteinase K–treated samples using a DNA purification kit following the manufacturer instructions (A9282; Promega) and was subsequently analyzed either by running a 2% low melting agarose (APEX) gel or by an Agilent Technologies 2100 Bioanalyzer by using the DNA 1000 kit.

    Techniques: In Vitro, Sedimentation, In Vivo, Expressing, Fractionation, Staining, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

    Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific PCR analysis of the chd gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.

    Journal: Scientific Reports

    Article Title: In vivo targeted single-nucleotide editing in zebrafish

    doi: 10.1038/s41598-018-29794-9

    Figure Lengend Snippet: Identification of heterozygous fish carrying the expected nucleotide substitution at the target site. ( A ) Schematic representation of allele-specific PCR analysis of the chd gene. ( B ) An example of the results after allele-specific PCR amplification of the chd gene. The 203 bp DNA fragments in the”w” lanes were amplified by the primer set of chd _wF and chd _wR primers, whereas fragments in the “m” lanes were amplified by the primer set chd _mF and chd _wR. The 635 bp DNA fragments were amplified by a primer set recognizing a different region of the chd gene as an internal control. ( C ) Results of analyzing all individuals by allele-specific PCR for the chd gene. “Negative” indicates the absence of a nucleotide substitution at the target site, while “positive” indicates the presence of a nucleotide substitution at the target site. ( D ) Results of analyzing 12 positive individuals by sequencing the targeted region of the chd gene. ( E ) Schematic representation of allele-specific PCR analysis for the oep gene. ( F ) An example of results after allele-specific PCR amplification of the oep gene. The 183 bp DNA fragments in the”w” lanes were amplified by the primer set oep _wF and oep _wR, whereas fragments in the “m” lanes were amplified by the primer set oep _wF and oep _mR. The 415 bp DNA fragments were amplified by a primer set recognizing a different region of the oep gene as an internal control. ( G ) Results of analyzing all individuals by allele-specific PCR for the oep gene. ( H ) Results of analyzing 16 positive individuals by sequencing the targeted region of the oep gene. ( D , H ) The red arrowheads indicate the nucleotide substitution at the target site. ( H ) The black arrowhead indicates the nucleotide substitution at the untargeted site.

    Article Snippet: The PCR products from the chd and oep amplifications were purified with NucleoSpin Gel and PCR Clean-up (Macherey-Nagel, Düren, Germany) and sequenced by Fasmac (Atsugi, Japan) or by Macrogen Japan (Kyoto, Japan) with the chd _1st_F and oep _1st_F primers, respectively.

    Techniques: Fluorescence In Situ Hybridization, Polymerase Chain Reaction, Amplification, Sequencing

    TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using PCR. b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses

    Journal: Epigenetics & Chromatin

    Article Title: Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination

    doi: 10.1186/s13072-018-0247-4

    Figure Lengend Snippet: TET1 and TET2 influence 5hmC on osteogenic genes. BMSC were cultured under normal growth conditions and treated with scramble siRNA or siRNA directed to TET1 (siTET1) or TET2 (siTET2) and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmC to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. a Relative enrichment of 5hmC on RUNX2 transcription start site (TSS) was measured using PCR. b 5hmC on BMP2 TSS was measured as in ( a ). BMSC were cultured under normal growth and genomic DNA purified and immunoprecipitated using an antibody to 5hmC. Recruitment of 5hmc to genomic regions was assessed by the hme-DIP analysis and normalised to the genomic input control. c Cells were cultured under normal and osteogenic conditions. Relative enrichment of 5hmC on RUNX2 transcription start site (TSS), exon and intron regions. d Relative enrichment of 5hmC on BMP2 TSS, exon and intron regions, percentage input. Data represent mean S.E.M., n = 3 BMSC donors, * p ≤ 0.05, one-way ANOVA with multiple comparison analyses

    Article Snippet: Immunoprecipitated bead–DNA complex was treated with proteinase K for 3 h at 50 °C in elution buffer and hydroxymethylated DNA purified by using the Qiagen PCR clean-up kit (Qiaquick).

    Techniques: Cell Culture, Purification, Immunoprecipitation, Polymerase Chain Reaction

    Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by PCR using primers specific to these regions (a). L-100 bp ladder. PCR products were sequenced by Sangers method. All the 4 nucleotide differences were also present at genomic DNA level. Representative image of exon specific amplification (b). Alignment of exon specific sequencing results with human, mouse, and rat (predicted) sequence shows 2 amino acid residue changes (c).

    Journal: BioMed Research International

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico

    doi: 10.1155/2017/5170680

    Figure Lengend Snippet: Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by PCR using primers specific to these regions (a). L-100 bp ladder. PCR products were sequenced by Sangers method. All the 4 nucleotide differences were also present at genomic DNA level. Representative image of exon specific amplification (b). Alignment of exon specific sequencing results with human, mouse, and rat (predicted) sequence shows 2 amino acid residue changes (c).

    Article Snippet: PCR amplified DNA was cleaned using PCR clean-up kit following the kit protocol (Promega, Wisconsin, USA), sequenced, and verified using nucleotide BLAST tool.

    Techniques: Amplification, Sequencing, Polymerase Chain Reaction

    Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.

    Journal: Frontiers in Microbiology

    Article Title: Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters

    doi: 10.3389/fmicb.2018.00830

    Figure Lengend Snippet: Pipeline of microfluidic nested PCR followed by MiSeq amplicon sequencing (MFnPCR–MiSeq). Purification of PCR amplicons was carried out with the NucleoSpin Gel and PCR Clean-up Kit. Amplification by the first-round and nested PCR was performed on a 48.48 Access Array (AA) chip in a BioMark HD reader, while the third PCR for addition of a sample-specific tag and Illumina adapter was run in 96-well PCR plates on a conventional thermal cycler.

    Article Snippet: 10 μL) were purified using NucleoSpin Gel and the PCR Clean-up Kit (Takara Bio, Shiga, Japan).

    Techniques: Nested PCR, Amplification, Sequencing, Purification, Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Rat floxed alleles generated by zygote electroporation. a Schematic representation of CLICK: CRISPR with lssDNA inducing conditional knockout alleles. b Schematic approach to generate rat Vapb floxed alleles using the CRISPR/Cas system with lssDNA composed of the targeted exon flanked by two loxP sites (Additional file 11 : Figure S11). c PCR analysis of rat pups (#1–6) generated by CLICK, showing different types of mutations, indels, LD (via NHEJ), inversions, and floxed alleles (via HDR) (black arrow). The primer sets were used for PCR and sequence analysis (Additional file 2 : Figure S2). M: 100 bp DNA ladder marker. d Sequence analysis of 6 pups showing a variety of mutations, indels, inversion or LD indicated by red letters, and loxP insertions and flox indicated by orange letters. All pups were used for testing germline transmission (Additional file 15 : Table S1). Asterisks indicate pups used for testing the Cre-loxP system (Additional file 17 : Table S3)

    Journal: BMC Genomics

    Article Title: CLICK: one-step generation of conditional knockout mice

    doi: 10.1186/s12864-018-4713-y

    Figure Lengend Snippet: Rat floxed alleles generated by zygote electroporation. a Schematic representation of CLICK: CRISPR with lssDNA inducing conditional knockout alleles. b Schematic approach to generate rat Vapb floxed alleles using the CRISPR/Cas system with lssDNA composed of the targeted exon flanked by two loxP sites (Additional file 11 : Figure S11). c PCR analysis of rat pups (#1–6) generated by CLICK, showing different types of mutations, indels, LD (via NHEJ), inversions, and floxed alleles (via HDR) (black arrow). The primer sets were used for PCR and sequence analysis (Additional file 2 : Figure S2). M: 100 bp DNA ladder marker. d Sequence analysis of 6 pups showing a variety of mutations, indels, inversion or LD indicated by red letters, and loxP insertions and flox indicated by orange letters. All pups were used for testing germline transmission (Additional file 15 : Table S1). Asterisks indicate pups used for testing the Cre-loxP system (Additional file 17 : Table S3)

    Article Snippet: Bands corresponding to a single-strand DNA fragment were extracted using NucleoSpin® Gel and PCR Clean-up (Takara Bio, Shiga, Japan).

    Techniques: Generated, Electroporation, CRISPR, Knock-Out, Polymerase Chain Reaction, Non-Homologous End Joining, Sequencing, Marker, Transmission Assay

    One-step generation of conditional knockout animals (F0) by CLICK. a Schematic representation of applying CLICK in oocytes for in vitro fertilization (IVF) with Cre-driver mice, Emx1-cre, resulting in brain-specific recombination at the targeted floxed alleles. b PCR analysis of representative delivered mouse pups by microinjection (#1–8) or electroporation (#9–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2 : Figure S2) were used for PCR and sequence analysis. c Genotyping in several tissues: cerebrum (Cr), cerebellum (Cl), heart (H), liver (L), spleen (S), and testis (Ts), indicating recombination (red arrow) by brain-specific Cre expression in #14 mouse carrying heterozygous floxed with LD alleles. M: 100 bp DNA ladder marker. d Time-schedule comparisons of targeting methods using ES cells by CRISPR in B6 oocytes, and CLICK in Cre oocytes. CLICK saves about 6 months of crossing and reduces breeding costs during the study period (Additional file 13 : Figure S13)

    Journal: BMC Genomics

    Article Title: CLICK: one-step generation of conditional knockout mice

    doi: 10.1186/s12864-018-4713-y

    Figure Lengend Snippet: One-step generation of conditional knockout animals (F0) by CLICK. a Schematic representation of applying CLICK in oocytes for in vitro fertilization (IVF) with Cre-driver mice, Emx1-cre, resulting in brain-specific recombination at the targeted floxed alleles. b PCR analysis of representative delivered mouse pups by microinjection (#1–8) or electroporation (#9–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2 : Figure S2) were used for PCR and sequence analysis. c Genotyping in several tissues: cerebrum (Cr), cerebellum (Cl), heart (H), liver (L), spleen (S), and testis (Ts), indicating recombination (red arrow) by brain-specific Cre expression in #14 mouse carrying heterozygous floxed with LD alleles. M: 100 bp DNA ladder marker. d Time-schedule comparisons of targeting methods using ES cells by CRISPR in B6 oocytes, and CLICK in Cre oocytes. CLICK saves about 6 months of crossing and reduces breeding costs during the study period (Additional file 13 : Figure S13)

    Article Snippet: Bands corresponding to a single-strand DNA fragment were extracted using NucleoSpin® Gel and PCR Clean-up (Takara Bio, Shiga, Japan).

    Techniques: Knock-Out, In Vitro, Mouse Assay, Polymerase Chain Reaction, Electroporation, Sequencing, Expressing, Marker, CRISPR

    Mouse floxed alleles generated by microinjection of two gRNA, Cas9 mRNA and lssDNA into zygotes. a An approach to generate Serpina3n floxed alleles using the CRISPR/Cas system with long single-stranded DNA (lssDNA) composed of the targeted exon flanked by two loxP sites (Additional file 2 : Figure S2). b PCR analysis of representative delivered mouse pups (#1–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2 : Figure S2) were used for PCR and sequence analysis. Asterisks indicate pups used for testing germline transmission (Additional file 15 : Table S1). M: 100 bp DNA ladder marker. c Representative examples of the targeted Serpina3n loci generated by the microinjection of two gRNA (gRNA-1 and gRNA-2), Cas9 mRNA and lssDNA into B6 mouse zygotes (Additional file 3 : Figure S3)

    Journal: BMC Genomics

    Article Title: CLICK: one-step generation of conditional knockout mice

    doi: 10.1186/s12864-018-4713-y

    Figure Lengend Snippet: Mouse floxed alleles generated by microinjection of two gRNA, Cas9 mRNA and lssDNA into zygotes. a An approach to generate Serpina3n floxed alleles using the CRISPR/Cas system with long single-stranded DNA (lssDNA) composed of the targeted exon flanked by two loxP sites (Additional file 2 : Figure S2). b PCR analysis of representative delivered mouse pups (#1–15) showing different types of mutations, indels, LD, and floxed alleles (black arrow) at the targeted Serpina3n locus. The primer sets (‘small’ in Additional file 2 : Figure S2) were used for PCR and sequence analysis. Asterisks indicate pups used for testing germline transmission (Additional file 15 : Table S1). M: 100 bp DNA ladder marker. c Representative examples of the targeted Serpina3n loci generated by the microinjection of two gRNA (gRNA-1 and gRNA-2), Cas9 mRNA and lssDNA into B6 mouse zygotes (Additional file 3 : Figure S3)

    Article Snippet: Bands corresponding to a single-strand DNA fragment were extracted using NucleoSpin® Gel and PCR Clean-up (Takara Bio, Shiga, Japan).

    Techniques: Generated, CRISPR, Polymerase Chain Reaction, Sequencing, Transmission Assay, Marker

    Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition

    Journal: Nature Communications

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice

    doi: 10.1038/s41467-019-13193-3

    Figure Lengend Snippet: Essential role of PRAMEF12 in spermatogonial differentiation. a Immunofluorescence of whole-mount testes from P2, P4, and P7 Pramef12 Null and Pramef12 Het mice after staining with antibodies to DDX4 (left) or KIT (middle) and merged (right). KIT-positive spermatogonia were rarely detected at P2 in either Pramef12 Null or Pramef12 Het tubules but were present at P4 and increased at P7 in Pramef12 Het tubules. Few KIT-positive spermatogonia (arrow) were observed in P7 Pramef12 Null tubules. Scale bar, 50 μm. b Immunohistochemistry of sections from P7 Pramef12 Null (top panels) and Pramef12 Het (bottom panels) testes after staining with antibodies to DDX4 (left) or KIT (middle) and merged with Hoechst 33342 to stain DNA (right). Arrowheads, KIT-positive cells in Pramef12 Null testes. Scale bar, 50 μm. c Quantitative real-time RT-PCR of Kit mRNA abundance in P7 Pramef12 Null and Pramef12 Het testes using β-actin as an internal load control and setting the abundance in Pramef12 Het testis to 1. Mean ± s.d, n = 3 biologically independent samples per condition. * P = 1.43E-05 by two-tailed Student’s t test. d Immunoblot of KIT protein in P7 Pramef12 Null and Pramef12 Het testes using α-tubulin as a load control. e Same as a , but with antibodies to SOHLH1, SOHLH2, and PLZF. The number of spermatogonia positive for SOHLH1 and SOHLH2 was significantly reduced in P7 Pramef12 Null tubules (top panels). Representative of n = 3 a , b , d , e independent biological replicates with similar results per condition

    Article Snippet: After linearization by digestion with DraI, the DNA fragment was purified with PCR Clean-up Kit (Clontech Laboratories) and transcribed using AmpliScribe T7-Flash Transcription Kit (Lucigen).

    Techniques: Immunofluorescence, Mouse Assay, Staining, Immunohistochemistry, Quantitative RT-PCR, Two Tailed Test

    Characterization of a stable eys rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.

    Journal: PLoS ONE

    Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish

    doi: 10.1371/journal.pone.0200789

    Figure Lengend Snippet: Characterization of a stable eys rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.

    Article Snippet: To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers.

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction

    Diagrammatic representation of the RNA silencing constructs (not drawn to scale). (A) A 21 nucleotide artificial microRNA (ami-BEIIb)-based osa -miR528 was synthesized by fusion PCR and cloned into Vec8, a Ti binary vector with a wheat high molecular weight glutenin promoter (wHMGPro) and nopaline synthase terminator (NOS). (B) The secondary structure of the osa -miR528 backbone as predicted by RNAfold, including information on ami-BEIIb (reverse complement). The predicted target cleavage site (arrow with sequence bold and highlighted) is located between positions 10 and 11 of the amiRNA, while the two mismatches (grey highlight) are located at positions 1 and 21. (C) The hairpin RNA (hp-BEIIb) was cloned in Vec8 by inserting a 397 bp BEIIb fragment in the sense (BEIIb→) and antisense (BEIIb←) orientations. The two fragments are flanked by two rice introns, Rint4 and Rint9, which form a hairpin loop. The amiRNA and hp-RNA fragments were directionally cloned using several restriction sites (H, Hin dIII; B, Bam HI; K, Kpn I; N, Not I; E Eco RI; S, Spe I).

    Journal: Journal of Experimental Botany

    Article Title: Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

    doi: 10.1093/jxb/err188

    Figure Lengend Snippet: Diagrammatic representation of the RNA silencing constructs (not drawn to scale). (A) A 21 nucleotide artificial microRNA (ami-BEIIb)-based osa -miR528 was synthesized by fusion PCR and cloned into Vec8, a Ti binary vector with a wheat high molecular weight glutenin promoter (wHMGPro) and nopaline synthase terminator (NOS). (B) The secondary structure of the osa -miR528 backbone as predicted by RNAfold, including information on ami-BEIIb (reverse complement). The predicted target cleavage site (arrow with sequence bold and highlighted) is located between positions 10 and 11 of the amiRNA, while the two mismatches (grey highlight) are located at positions 1 and 21. (C) The hairpin RNA (hp-BEIIb) was cloned in Vec8 by inserting a 397 bp BEIIb fragment in the sense (BEIIb→) and antisense (BEIIb←) orientations. The two fragments are flanked by two rice introns, Rint4 and Rint9, which form a hairpin loop. The amiRNA and hp-RNA fragments were directionally cloned using several restriction sites (H, Hin dIII; B, Bam HI; K, Kpn I; N, Not I; E Eco RI; S, Spe I).

    Article Snippet: After PCR purification using Wizard SV PCR Clean-up System (Promega), the amiRNA precursor (254 bp) was cloned into pGEM-T Easy (Promega) using E. coli DH5α.

    Techniques: Construct, Synthesized, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Molecular Weight, Sequencing