Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Organisation of the Gnas cluster. First exons of protein coding transcripts are shown as filled boxes and first exons of noncoding transcripts as shaded boxes. Arrows show the initiation and direction of transcription. Nesp is transcribed across the cluster. The arrow corresponding to the paternal Gnas allele is shown as a dotted line to indicate that the paternal Gnas allele is repressed tissue specifically. The 10 kb and 12.6 kb lines represent the new truncation mutations Nespas-T int2 and Nespas-T int3 . Nespas non-coding transcripts are shown as grey lines. * represents the termination of Nespas transcription, approximately 30 kb from the Nespas promoter. Paternally expressed microRNAs are shown as vertical lines. Filled circles represent methylated regions. ICR represents the imprinting control region.

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Methylation, Control

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Truncation of Nespas before the Nesp promoter in Nespas intron 2/ Nesp exon 2. ( a ) Schematic of the mouse Gnas locus showing the site of insertion of the rabbit β-globin polyadenylation cassette . The targeting vector shows the location of a 1.2 kb fragment (black box labelled Ap) from the rabbit β-globin gene to truncate Nespas and the position of the selection cassette (TKneopA URA3), flanked by loxP sites (open triangles). The targeted allele was designated Nespas-T int2 allele after Cre-mediated excision of the selectable marker cassette. Methylated DMRs are shown by filled circles. A, Afl II; X, Xho I; ICR, Imprinting Control Region. The arrow shows the approximate position of the polyadenylation cassette (pA) to truncate Nespas in exon 1 in Nespas-T ex1 ; ( b ) Southern analysis of ES cell DNA showing correct targeting in the Ap- neo allele. Ap- neo targeted clones were identified by the presence of a 12.0 kb Afl II fragment detected with the 5' external probe, probe A. Correct targeting at the 3' end was confirmed by the detection of a 14.2 kb Xho I fragment with Probe B; ( c ) Nespas is downregulated after the insertion; ( d ) Nespas is upregulated before the insertion; ( e ) Nespas is truncated and abundant in +/ Nespas-T int2 ; ( f ) Bisulphite analysis showing the Nesp DMR is unmethylated on the paternal allele in + SD2 / Nespas-T int2 . All clones from one individual are grouped into a block and two + SD2 / Nespas-T int2 individuals, (1) and (2), and one + SD2 /+ individual were analysed. Each row of circles represents a clone derived from the paternal allele and each circle corresponds to a separate CpG (filled circle, methylated CpG; unfilled circle, unmethylated CpG) for nucleotides 140,450–140,800; ( g ) The Nesp level is unaffected in +/ Nespas-T int2 .

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Plasmid Preparation, Selection, Marker, Methylation, Control, Clone Assay, Blocking Assay, Derivative Assay

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Transcription and methylation status of the Gnas cluster is unaltered on paternal inheritance of Nesp trun . ( a ) Schematic summary of the transcriptional and methylation status of Nesp and Nespas on the paternal allele in wild-type and +/ Nesp trun . The pA insertion will truncate Nesp if expressed. Row of filled circles, methylated allele; row of unfilled circles, unmethylated allele; ( b ) The level of Nespas exon 1 spliced onto exon 2 is unaltered in +/ Nesp trun ; ( c ) Methylation at the Nesp DMR is unaffected. PCR products from bisulphite-treated DNAs were separated on 3% agarose gels as either undigested (U) or digested with Taq 1 (T). Digestion products in the T lanes represent methylation of the CpG dinucleotide in the Taq 1 recognition sequence. Genomic DNA from +/ Nespas-T int2 in which both alleles were unmethylated was included as a negative control; ( d ) The levels of Nesp , Gnasxl and Exon1A ( Ex1A ) are unaffected.

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Methylation, Sequencing, Negative Control

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Truncation of Nespas after the Nesp promoter in Nespas intron 4. ( a ) Schematic of the mouse Gnas locus showing the site of insertion of the rabbit β-globin polyadenylation cassette (black box labelled Ap) to truncate Nespas and the position of the selection cassette flanked by loxP sites (open triangles). The selection cassette was deleted on germline transmission and the targeted allele was designated Nespas-T int3 . A, Afl II; B, Bsr GI; X, Xho I; ( b ) Southern analysis of ES cell DNA showing correct targeting. Correctly targeted clones were identified by the presence of a 14.1 kb Afl II fragment detected with the 5′ external probe, probe A. Correct targeting at the 3′ end was confirmed by the detection of a 16.3 kb Bsr GI fragment with probe B; ( c ) Nespas is downregulated after the insertion; ( d ) Nespas is upregulated before the insertion. There was a greater fold difference in the spliced form (exon 1 spliced onto exon 2) than the unspliced form (intron 4); ( e ) Nespas is truncated and abundant in +/ Nespas-T int3 ; ( f ) Northern blot assay of Nespas and loading control Actb ; ( g ) Light Scanner melt profile, using a SNP in Nespas exon 3, to show Nespas is expressed from the paternal allele in +/ Nespas-T int3 ; ( h ) Bisulphite analysis showing the Nesp DMR is unmethylated on the maternal allele and methylated on the paternal allele in + SD2 / NespasT int3 ; ( i ) The Nesp level is unaffected in +/ Nespas-T int3 .

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Selection, Transmission Assay, Clone Assay, Northern Blot, Control, Methylation

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Levels of Gnasxl , Exon1A and Gnas in + / Nespas-T int2 and +/ Nespas-T int3 . ( a ) and ( b ) Gnasxl is downregulated in +/ Nespas-T int2 whereas Exon1A ( Ex1A ) and Gnas are unaffected; ( c ) and ( d ) Gnasxl , Exon1A and Gnas are unaffected in +/ Nespas-T int3 . Gnasxl and Ex1A levels were measured in newborn brain whereas Gnas and Ex1A were analysed in brown fat, a tissue in which Gnas is predominantly expressed from the maternal allele [ , ].

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques:

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Summary of the transcriptional and methylation status of the paternal allele of the imprinted Gnas cluster in wild-type and mutant mice. ( a ) wild-type; ( b ) +/ Nespas-T ex1 ; ( c ) +/ Nespas-T int2 ; ( d ) +/ Nesp trun ; ( e ) +/ Nespas-T int3 . The Ap insertion truncates Nespas and the pA insertion will truncate Nesp if expressed. The approximate positions of the differentially methylated regions (DMRs) are shown by rows of filled circles on the methylated allele and unfilled circles on the unmethylated allele. Arrows show the start and direction of transcription, with thin arrows indicating weak transcription. Multiple arrows in ( c ) and ( e ) show increased levels of truncated Nespas . Exons 2–12 of Gnas are not shown. The figure is not drawn to scale. Information for ( b ), +/ Nespas-T ex1 , taken from [ , ].

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Methylation, Mutagenesis

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Organisation of the Gnas cluster. First exons of protein coding transcripts are shown as filled boxes and first exons of noncoding transcripts as shaded boxes. Arrows show the initiation and direction of transcription. Nesp is transcribed across the cluster. The arrow corresponding to the paternal Gnas allele is shown as a dotted line to indicate that the paternal Gnas allele is repressed tissue specifically. The 10 kb and 12.6 kb lines represent the new truncation mutations Nespas-T int2 and Nespas-T int3 . Nespas non-coding transcripts are shown as grey lines. * represents the termination of Nespas transcription, approximately 30 kb from the Nespas promoter. Paternally expressed microRNAs are shown as vertical lines. Filled circles represent methylated regions. ICR represents the imprinting control region.

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Methylation, Control

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Truncation of Nespas before the Nesp promoter in Nespas intron 2/ Nesp exon 2. ( a ) Schematic of the mouse Gnas locus showing the site of insertion of the rabbit β-globin polyadenylation cassette . The targeting vector shows the location of a 1.2 kb fragment (black box labelled Ap) from the rabbit β-globin gene to truncate Nespas and the position of the selection cassette (TKneopA URA3), flanked by loxP sites (open triangles). The targeted allele was designated Nespas-T int2 allele after Cre-mediated excision of the selectable marker cassette. Methylated DMRs are shown by filled circles. A, Afl II; X, Xho I; ICR, Imprinting Control Region. The arrow shows the approximate position of the polyadenylation cassette (pA) to truncate Nespas in exon 1 in Nespas-T ex1 ; ( b ) Southern analysis of ES cell DNA showing correct targeting in the Ap- neo allele. Ap- neo targeted clones were identified by the presence of a 12.0 kb Afl II fragment detected with the 5' external probe, probe A. Correct targeting at the 3' end was confirmed by the detection of a 14.2 kb Xho I fragment with Probe B; ( c ) Nespas is downregulated after the insertion; ( d ) Nespas is upregulated before the insertion; ( e ) Nespas is truncated and abundant in +/ Nespas-T int2 ; ( f ) Bisulphite analysis showing the Nesp DMR is unmethylated on the paternal allele in + SD2 / Nespas-T int2 . All clones from one individual are grouped into a block and two + SD2 / Nespas-T int2 individuals, (1) and (2), and one + SD2 /+ individual were analysed. Each row of circles represents a clone derived from the paternal allele and each circle corresponds to a separate CpG (filled circle, methylated CpG; unfilled circle, unmethylated CpG) for nucleotides 140,450–140,800; ( g ) The Nesp level is unaffected in +/ Nespas-T int2 .

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Plasmid Preparation, Selection, Marker, Methylation, Control, Clone Assay, Blocking Assay, Derivative Assay

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Transcription and methylation status of the Gnas cluster is unaltered on paternal inheritance of Nesp trun . ( a ) Schematic summary of the transcriptional and methylation status of Nesp and Nespas on the paternal allele in wild-type and +/ Nesp trun . The pA insertion will truncate Nesp if expressed. Row of filled circles, methylated allele; row of unfilled circles, unmethylated allele; ( b ) The level of Nespas exon 1 spliced onto exon 2 is unaltered in +/ Nesp trun ; ( c ) Methylation at the Nesp DMR is unaffected. PCR products from bisulphite-treated DNAs were separated on 3% agarose gels as either undigested (U) or digested with Taq 1 (T). Digestion products in the T lanes represent methylation of the CpG dinucleotide in the Taq 1 recognition sequence. Genomic DNA from +/ Nespas-T int2 in which both alleles were unmethylated was included as a negative control; ( d ) The levels of Nesp , Gnasxl and Exon1A ( Ex1A ) are unaffected.

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Methylation, Sequencing, Negative Control

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Truncation of Nespas after the Nesp promoter in Nespas intron 4. ( a ) Schematic of the mouse Gnas locus showing the site of insertion of the rabbit β-globin polyadenylation cassette (black box labelled Ap) to truncate Nespas and the position of the selection cassette flanked by loxP sites (open triangles). The selection cassette was deleted on germline transmission and the targeted allele was designated Nespas-T int3 . A, Afl II; B, Bsr GI; X, Xho I; ( b ) Southern analysis of ES cell DNA showing correct targeting. Correctly targeted clones were identified by the presence of a 14.1 kb Afl II fragment detected with the 5′ external probe, probe A. Correct targeting at the 3′ end was confirmed by the detection of a 16.3 kb Bsr GI fragment with probe B; ( c ) Nespas is downregulated after the insertion; ( d ) Nespas is upregulated before the insertion. There was a greater fold difference in the spliced form (exon 1 spliced onto exon 2) than the unspliced form (intron 4); ( e ) Nespas is truncated and abundant in +/ Nespas-T int3 ; ( f ) Northern blot assay of Nespas and loading control Actb ; ( g ) Light Scanner melt profile, using a SNP in Nespas exon 3, to show Nespas is expressed from the paternal allele in +/ Nespas-T int3 ; ( h ) Bisulphite analysis showing the Nesp DMR is unmethylated on the maternal allele and methylated on the paternal allele in + SD2 / NespasT int3 ; ( i ) The Nesp level is unaffected in +/ Nespas-T int3 .

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Selection, Transmission Assay, Clone Assay, Northern Blot, Control, Methylation

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Levels of Gnasxl , Exon1A and Gnas in + / Nespas-T int2 and +/ Nespas-T int3 . ( a ) and ( b ) Gnasxl is downregulated in +/ Nespas-T int2 whereas Exon1A ( Ex1A ) and Gnas are unaffected; ( c ) and ( d ) Gnasxl , Exon1A and Gnas are unaffected in +/ Nespas-T int3 . Gnasxl and Ex1A levels were measured in newborn brain whereas Gnas and Ex1A were analysed in brown fat, a tissue in which Gnas is predominantly expressed from the maternal allele [ , ].

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques:

Journal: Non-Coding RNA

Article Title: Antisense Activity across the Nesp Promoter is Required for Nespas -Mediated Silencing in the Imprinted Gnas Cluster

doi: 10.3390/ncrna1030246

Figure Lengend Snippet: Summary of the transcriptional and methylation status of the paternal allele of the imprinted Gnas cluster in wild-type and mutant mice. ( a ) wild-type; ( b ) +/ Nespas-T ex1 ; ( c ) +/ Nespas-T int2 ; ( d ) +/ Nesp trun ; ( e ) +/ Nespas-T int3 . The Ap insertion truncates Nespas and the pA insertion will truncate Nesp if expressed. The approximate positions of the differentially methylated regions (DMRs) are shown by rows of filled circles on the methylated allele and unfilled circles on the unmethylated allele. Arrows show the start and direction of transcription, with thin arrows indicating weak transcription. Multiple arrows in ( c ) and ( e ) show increased levels of truncated Nespas . Exons 2–12 of Gnas are not shown. The figure is not drawn to scale. Information for ( b ), +/ Nespas-T ex1 , taken from [ , ].

Article Snippet: Other assays were for Gnasxl (Mm01717466_g1), Ex1A (Mm01248152_m1), and Gnas (Mm00507037_m1).

Techniques: Methylation, Mutagenesis