Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation
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{"title"=>"Reporter gene silencing in targeted mouse mutants is associated with promoter CpG island methylation", "type"=>"journal", "authors"=>[{"first_name"=>"Julia V.", "last_name"=>"Kirov", "scopus_author_id"=>"56884815600"}, {"first_name"=>"Michael", "last_name"=>"Adkisson", "scopus_author_id"=>"6506161673"}, {"first_name"=>"A. J.", "last_name"=>"Nava", "scopus_author_id"=>"56884090200"}, {"first_name"=>"Andreana", "last_name"=>"Cipollone", "scopus_author_id"=>"56884457700"}, {"first_name"=>"Brandon", "last_name"=>"Willis", "scopus_author_id"=>"56720917800"}, {"first_name"=>"Eric K.", "last_name"=>"Engelhard", "scopus_author_id"=>"6602849900"}, {"first_name"=>"K. C.Kent", "last_name"=>"Lloyd", "scopus_author_id"=>"35230265300"}, {"first_name"=>"Pieter", "last_name"=>"De Jong", "scopus_author_id"=>"55658056927"}, {"first_name"=>"David B.", "last_name"=>"West", "scopus_author_id"=>"7401998228"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "sgr"=>"84942898264", "isbn"=>"10.1371/journal.pone.0134155", "doi"=>"10.1371/journal.pone.0134155", "pui"=>"606195336", "pmid"=>"26275310", "scopus"=>"2-s2.0-84942898264"}, "id"=>"c1816375-d83e-327c-8dce-a8b1ab277b2e", "abstract"=>"Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfitetreated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.", "link"=>"http://www.mendeley.com/research/reporter-gene-silencing-targeted-mouse-mutants-associated-promoter-cpg-island-methylation", "reader_count"=>8, "reader_count_by_academic_status"=>{"Researcher"=>3, "Student > Ph. D. Student"=>2, "Student > Master"=>1, "Other"=>1, "Student > Bachelor"=>1}, "reader_count_by_user_role"=>{"Researcher"=>3, "Student > Ph. D. Student"=>2, "Student > Master"=>1, "Other"=>1, "Student > Bachelor"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>2, "Medicine and Dentistry"=>2, "Agricultural and Biological Sciences"=>4}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>4}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}}, "reader_count_by_country"=>{"South Africa"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2212843"], "description"=>"<p>A cartoon of the knockout first allele is shown with exons indicated by gray blocks numbered 1–3. The targeted exon in this schematic is #2, with the targeting vector replacing that critical exon with an identical exon flanked with LoxP sites. Proximal to the critical exon are placed in tandem a LacZ reporter gene driven by the targeted gene promoter, followed by transcriptional stop and polyadenylation signal, and then followed by a heterologous promoter driving a neomycin resistance gene. The gene regions evaluated for expression by qRT-PCR and/or for methylation by sequencing bisulfite treated DNA are indicated in brackets.</p>", "links"=>[], "tags"=>["chromatin structure", "LacZ coding sequence methylation", "CpG island methylation", "reporter genes", "reporter gene", "gene promoter", "Silenced mutants", "reporter Gene Silencing", "Targeted Mouse Mutants", "selection cassette", "results support", "Promoter CpG Island Methylation Targeted mutations", "messenger RNA levels", "tm 1a Knockout Mouse Project allele", "dna", "promoter CpG methylation", "LacZ reporter gene", "promoter CpG islands", "quantities LacZ mRNA", "LacZ staining", "tm 1a allele", "LacZ gene mRNA", "LacZ coding sequence"], "article_id"=>1511264, "categories"=>["Biological Sciences"], "users"=>["Julia V. Kirov", "Michael Adkisson", "A. J. Nava", "Andreana Cipollone", "Brandon Willis", "Eric K. Engelhard", "K. C. Kent Lloyd", "Pieter de Jong", "David B. West"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0134155.g001", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_of_Experimental_Approach_/1511264", "title"=>"Schematic of Experimental Approach.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-08-14 03:04:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/2212855", "https://ndownloader.figshare.com/files/2212856", "https://ndownloader.figshare.com/files/2212857", "https://ndownloader.figshare.com/files/2212858", "https://ndownloader.figshare.com/files/2212859", "https://ndownloader.figshare.com/files/2212860", "https://ndownloader.figshare.com/files/2212861", "https://ndownloader.figshare.com/files/2212862", "https://ndownloader.figshare.com/files/2212863", "https://ndownloader.figshare.com/files/2212864", "https://ndownloader.figshare.com/files/2212865"], "description"=>"<div><p>Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.</p></div>", "links"=>[], "tags"=>["chromatin structure", "LacZ coding sequence methylation", "CpG island methylation", "reporter genes", "reporter gene", "gene promoter", "Silenced mutants", "reporter Gene Silencing", "Targeted Mouse Mutants", "selection cassette", "results support", "Promoter CpG Island Methylation Targeted mutations", "messenger RNA levels", "tm 1a Knockout Mouse Project allele", "dna", "promoter CpG methylation", "LacZ reporter gene", "promoter CpG islands", "quantities LacZ mRNA", "LacZ staining", "tm 1a allele", "LacZ gene mRNA", "LacZ coding sequence"], "article_id"=>1511276, "categories"=>["Biological Sciences"], "users"=>["Julia V. Kirov", "Michael Adkisson", "A. J. Nava", "Andreana Cipollone", "Brandon Willis", "Eric K. Engelhard", "K. C. Kent Lloyd", "Pieter de Jong", "David B. West"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0134155.s001", "https://dx.doi.org/10.1371/journal.pone.0134155.s002", "https://dx.doi.org/10.1371/journal.pone.0134155.s003", "https://dx.doi.org/10.1371/journal.pone.0134155.s004", "https://dx.doi.org/10.1371/journal.pone.0134155.s005", "https://dx.doi.org/10.1371/journal.pone.0134155.s006", "https://dx.doi.org/10.1371/journal.pone.0134155.s007", "https://dx.doi.org/10.1371/journal.pone.0134155.s008", "https://dx.doi.org/10.1371/journal.pone.0134155.s009", "https://dx.doi.org/10.1371/journal.pone.0134155.s010", "https://dx.doi.org/10.1371/journal.pone.0134155.s011"], "stats"=>{"downloads"=>15, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Reporter_Gene_Silencing_in_Targeted_Mouse_Mutants_Is_Associated_with_Promoter_CpG_Island_Methylation_/1511276", "title"=>"Reporter Gene Silencing in Targeted Mouse Mutants Is Associated with Promoter CpG Island Methylation", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-08-14 03:04:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/2212852"], "description"=>"<p>CpG Island Primers.</p>", "links"=>[], "tags"=>["chromatin structure", "LacZ coding sequence methylation", "CpG island methylation", "reporter genes", "reporter gene", "gene promoter", "Silenced mutants", "reporter Gene Silencing", "Targeted Mouse Mutants", "selection cassette", "results support", "Promoter CpG Island Methylation Targeted mutations", "messenger RNA levels", "tm 1a Knockout Mouse Project allele", "dna", "promoter CpG methylation", "LacZ reporter gene", "promoter CpG islands", "quantities LacZ mRNA", "LacZ staining", "tm 1a allele", "LacZ gene mRNA", "LacZ coding sequence"], "article_id"=>1511273, "categories"=>["Biological Sciences"], "users"=>["Julia V. Kirov", "Michael Adkisson", "A. J. Nava", "Andreana Cipollone", "Brandon Willis", "Eric K. Engelhard", "K. C. Kent Lloyd", "Pieter de Jong", "David B. West"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0134155.t002", "stats"=>{"downloads"=>3, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CpG_Island_Primers_/1511273", "title"=>"CpG Island Primers.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2015-08-14 03:04:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/2212850"], "description"=>"<p>% methylation of CpGs in the LacZ coding sequence is presented for each mutant and tissue, with Silenced mutants in red and Expressed mutants in blue. There was a significant overall difference in average percent methylation of LacZ coding sequence between the two groups (t-test; p < 0.001).</p>", "links"=>[], "tags"=>["chromatin structure", "LacZ coding sequence methylation", "CpG island methylation", "reporter genes", "reporter gene", "gene promoter", "Silenced mutants", "reporter Gene Silencing", "Targeted Mouse Mutants", "selection cassette", "results support", "Promoter CpG Island Methylation Targeted mutations", "messenger RNA levels", "tm 1a Knockout Mouse Project allele", "dna", "promoter CpG methylation", "LacZ reporter gene", "promoter CpG islands", "quantities LacZ mRNA", "LacZ staining", "tm 1a allele", "LacZ gene mRNA", "LacZ coding sequence"], "article_id"=>1511271, "categories"=>["Biological Sciences"], "users"=>["Julia V. Kirov", "Michael Adkisson", "A. J. Nava", "Andreana Cipollone", "Brandon Willis", "Eric K. Engelhard", "K. C. Kent Lloyd", "Pieter de Jong", "David B. West"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0134155.g006", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Methylation_of_CpGs_in_LacZ_Coding_Sequence_/1511271", "title"=>"Methylation of CpGs in LacZ Coding Sequence.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-08-14 03:04:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/2212851"], "description"=>"<p>qRT-PCR Primers and Probes.</p>", "links"=>[], "tags"=>["chromatin structure", "LacZ coding sequence methylation", "CpG island methylation", "reporter genes", "reporter gene", "gene promoter", "Silenced mutants", "reporter Gene Silencing", "Targeted Mouse Mutants", "selection cassette", "results support", "Promoter CpG Island Methylation Targeted mutations", "messenger RNA levels", "tm 1a Knockout Mouse Project allele", "dna", "promoter CpG methylation", "LacZ reporter gene", "promoter CpG islands", "quantities LacZ mRNA", "LacZ staining", "tm 1a allele", "LacZ gene mRNA", "LacZ coding sequence"], "article_id"=>1511272, "categories"=>["Biological Sciences"], "users"=>["Julia V. Kirov", "Michael Adkisson", "A. J. Nava", "Andreana Cipollone", "Brandon Willis", "Eric K. Engelhard", "K. C. Kent Lloyd", "Pieter de Jong", "David B. West"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0134155.t001", "stats"=>{"downloads"=>0, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_qRT_PCR_Primers_and_Probes_/1511272", "title"=>"qRT-PCR Primers and Probes.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2015-08-14 03:04:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/2212849"], "description"=>"<p>A significant positive correlation was observed between LacZ and CpG % methylation with a p < 0.013. Although the correlation between CpG% methylation and LacZ gene expression was negative as expected, this did not reach statistical significance.</p>", "links"=>[], "tags"=>["chromatin structure", "LacZ coding sequence methylation", "CpG island methylation", "reporter genes", "reporter gene", "gene promoter", "Silenced mutants", "reporter Gene Silencing", "Targeted Mouse Mutants", "selection cassette", "results support", "Promoter CpG Island Methylation Targeted mutations", "messenger RNA levels", "tm 1a Knockout Mouse Project allele", "dna", "promoter CpG methylation", "LacZ reporter gene", "promoter CpG islands", "quantities LacZ mRNA", "LacZ staining", "tm 1a allele", "LacZ gene mRNA", "LacZ coding sequence"], "article_id"=>1511270, "categories"=>["Biological Sciences"], "users"=>["Julia V. Kirov", "Michael Adkisson", "A. J. Nava", "Andreana Cipollone", "Brandon Willis", "Eric K. Engelhard", "K. C. Kent Lloyd", "Pieter de Jong", "David B. West"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0134155.g005", "stats"=>{"downloads"=>0, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Correlation_between_Promoter_and_LacZ_Coding_Sequence_Methylation_and_between_Promoter_Methylation_and_LacZ_Expression_in_Silenced_Mutants_/1511270", "title"=>"Correlation between Promoter and LacZ Coding Sequence Methylation, and between Promoter Methylation and LacZ Expression in Silenced Mutants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-08-14 03:04:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/2212846"], "description"=>"<p>Percent methylation of CpG islands (CGI) in Mutant tissues and in Wild Type controls are presented for the Silenced and Expressed groups. Although the differences were small for some CGIs the Cochran-Mantel-Haenszel test determined that each mutant CpG island was significantly different from the corresponding Wild Type Control island percent methylation.</p>", "links"=>[], "tags"=>["chromatin structure", "LacZ coding sequence methylation", "CpG island methylation", "reporter genes", "reporter gene", "gene promoter", "Silenced mutants", "reporter Gene Silencing", "Targeted Mouse Mutants", "selection cassette", "results support", "Promoter CpG Island Methylation Targeted mutations", "messenger RNA levels", "tm 1a Knockout Mouse Project allele", "dna", "promoter CpG methylation", "LacZ reporter gene", "promoter CpG islands", "quantities LacZ mRNA", "LacZ staining", "tm 1a allele", "LacZ gene mRNA", "LacZ coding sequence"], "article_id"=>1511267, "categories"=>["Biological Sciences"], "users"=>["Julia V. Kirov", "Michael Adkisson", "A. J. Nava", "Andreana Cipollone", "Brandon Willis", "Eric K. Engelhard", "K. C. Kent Lloyd", "Pieter de Jong", "David B. West"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0134155.g003", "stats"=>{"downloads"=>2, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Methylation_of_CpG_Islands_in_Targeted_Genes_/1511267", "title"=>"Methylation of CpG Islands in Targeted Genes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-08-14 03:04:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/2212845"], "description"=>"<p>Silenced and Expressed group targeted gene expression, and LacZ reporter gene expression, are presented as Relative Quantity (2<sup>-ΔΔCt</sup>) values relative to native gene expression in Wild Type Control animals for each mutant gene and tissue. Each data point is the average of three biological replicates, with the qPCR done in triplicate.</p>", "links"=>[], "tags"=>["chromatin structure", "LacZ coding sequence methylation", "CpG island methylation", "reporter genes", "reporter gene", "gene promoter", "Silenced mutants", "reporter Gene Silencing", "Targeted Mouse Mutants", "selection cassette", "results support", "Promoter CpG Island Methylation Targeted mutations", "messenger RNA levels", "tm 1a Knockout Mouse Project allele", "dna", "promoter CpG methylation", "LacZ reporter gene", "promoter CpG islands", "quantities LacZ mRNA", "LacZ staining", "tm 1a allele", "LacZ gene mRNA", "LacZ coding sequence"], "article_id"=>1511266, "categories"=>["Biological Sciences"], "users"=>["Julia V. Kirov", "Michael Adkisson", "A. J. Nava", "Andreana Cipollone", "Brandon Willis", "Eric K. Engelhard", "K. C. Kent Lloyd", "Pieter de Jong", "David B. West"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0134155.g002", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_RQ_mRNA_of_Targeted_Genes_and_LacZ_/1511266", "title"=>"RQ mRNA of Targeted Genes and LacZ.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-08-14 03:04:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/2212853"], "description"=>"<p>LacZ primers used for Amplifying Reporter Gene Sequence.</p>", "links"=>[], "tags"=>["chromatin structure", "LacZ coding sequence methylation", "CpG island methylation", "reporter genes", "reporter gene", "gene promoter", "Silenced mutants", "reporter Gene Silencing", "Targeted Mouse Mutants", "selection cassette", "results support", "Promoter CpG Island Methylation Targeted mutations", "messenger RNA levels", "tm 1a Knockout Mouse Project allele", "dna", "promoter CpG methylation", "LacZ reporter gene", "promoter CpG islands", "quantities LacZ mRNA", "LacZ staining", "tm 1a allele", "LacZ gene mRNA", "LacZ coding sequence"], "article_id"=>1511274, "categories"=>["Biological Sciences"], "users"=>["Julia V. Kirov", "Michael Adkisson", "A. J. Nava", "Andreana Cipollone", "Brandon Willis", "Eric K. Engelhard", "K. C. Kent Lloyd", "Pieter de Jong", "David B. West"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0134155.t003", "stats"=>{"downloads"=>0, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_LacZ_primers_used_for_Amplifying_Reporter_Gene_Sequence_/1511274", "title"=>"LacZ primers used for Amplifying Reporter Gene Sequence.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2015-08-14 03:04:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/2212847"], "description"=>"<p>Individual mutant and tissue values were averaged across the Silenced group (n = 7) and the Expressed group (n = 18) and compared by t-test. * = p<0.01; ** = p<0.0001. Date are presented as means +/- standard errors. The Silenced group had significantly lower expression of the targeted gene and the LacZ reporter and had higher percent methylation compared with the Expressed group.</p>", "links"=>[], "tags"=>["chromatin structure", "LacZ coding sequence methylation", "CpG island methylation", "reporter genes", "reporter gene", "gene promoter", "Silenced mutants", "reporter Gene Silencing", "Targeted Mouse Mutants", "selection cassette", "results support", "Promoter CpG Island Methylation Targeted mutations", "messenger RNA levels", "tm 1a Knockout Mouse Project allele", "dna", "promoter CpG methylation", "LacZ reporter gene", "promoter CpG islands", "quantities LacZ mRNA", "LacZ staining", "tm 1a allele", "LacZ gene mRNA", "LacZ coding sequence"], "article_id"=>1511268, "categories"=>["Biological Sciences"], "users"=>["Julia V. Kirov", "Michael Adkisson", "A. J. Nava", "Andreana Cipollone", "Brandon Willis", "Eric K. Engelhard", "K. C. Kent Lloyd", "Pieter de Jong", "David B. West"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0134155.g004", "stats"=>{"downloads"=>4, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Targeted_Gene_Expression_Reporter_Gene_Expression_and_Promoter_Methylation_/1511268", "title"=>"Targeted Gene Expression, Reporter Gene Expression and Promoter Methylation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-08-14 03:04:53"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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