Udies using this gene in a similar manner [31,32], Ppia showed no sign of circadian or ultradian variation (data not shown). The comparative CT method was similarly applied to determine the relative expression of Bmal1 mRNA. The following primers were used for real-time PCR analysis: Bmal1 forward: 59- CCAAGAAAGTATGGACACAGACAAA39; Bmal1 reverse: 59- GCATTCTTGATCCTTCCTTGGT-39; Ppia forward: 59- TGTGCCAGGGTGGTGACTT-39; Ppia reverse: 59- TCAAATTTCTCTCCGTAGATGGACTT39.Experiment 2: Mutagenic Analysis of Putative Binding Sites Mediating miR-142-3p-induced Repression of Bmal1 39 UTR ActivitymiTargetTM miRNA Target Sequence 39 UTR Expression Clone containing Bmal1 39 UTR sequence (Accession: NM_007489.3) inserted in the pEZX-MT01 vector was purchased from GeneCopoeia, Inc (Rockville, MD). The plasmid was propagated using methods Title Loaded From File established in our previous study [26]. Deletions in predicted miR-142-3p binding sites on the Bmal1 16574785 39 UTR were generated using QuikChange II XL SiteDirected Mutagenesis Kit (Stratagene, La Jolla, CA) Title Loaded From File according to the manufacturer’s protocols. Briefly, the full-length Bmal1 39 UTR was PCR mutagenized using specific primers so as to delete nucleotides 1? complementary to miR-142-3p seed region. After Dnp1-mediated degradation of parental plasmid DNA, the mutagenized plasmid was transformed into XL10-Gold Ultracompetent cells (Stratagene) and transformants were selected on kanamycin-containing (final conc. = 50 mg/ml) imMedia agar plates (Invitrogen). A single colony was isolated and propagated in imMedia Kan+ liquid medium (final conc. = 50 mg/ml). TheBmal1 constructs.UTRluciferasereportermiR-142-3p Modulation of BMAL1 in SCN Pacemakerplasmid was extracted using HiSpeed Plasmid Midi Kit (Qiagen, Inc.) and then sequenced to verify the targeted deletion (Bmal1 c.1_7del). Identical methods were also used to delete nucleotides 335?57 corresponding to a second predicted miR-142-3p binding site on the Bmal1 39 UTR complementary to the seed region along with additional nucleotides that may function as a 39 supplementary or compensatory element and aid in miRNA biological activity [33?5]. The resulting plasmid (Bmal1 c.335_357del) was then subjected to a second round of mutagenesis to provide for additional deletion of nucleotides 1?. The plasmid with targeted deletions of both miR-142-3p target sites on the Bmal1 39 UTR (Bmal1 c.1_7del+c.335_357del) was propagated and sequenced to verify these deletions as described above. The miRNA 39 UTR target control vector (Genecopoeia; CmiT000001-MT01), consisting of the pEZX-MT01 vector backbone without any 39 UTR sequence, was used to determine the specificity of miRNA interactions with the full-length and mutagenized Bmal1 vectors. miR-142-3p-mediated regulation of Bmal1 39 UTR was analyzed in human embryonic kidney (HEK293) cells using established methods [26]. Briefly, cells were seeded onto 24-well plates (Corning, Inc., Tewksbury MA) and 24 h later were co-transfected with pEZX-MR04 miR-142 expression vector (miExpress Precursor miRNA expression clone; Genecopoeia, Inc., MmiR3437MR04) and with either the target control, full-length Bmal1 39 UTR (WT), Bmal1 23977191 c.1_7del, Bmal1 c.335_357del or Bmal1 c.1_7del+c.335_357del miRNA 39 UTR target clones. As an additional control for specificity of the deletion, cells were also co-transfected with miR-494 expression vector and either the target control, Bmal1 or Bmal1 c.1_7del+c.335_357del miRNA 39 UTR target clones. Following transfection for.Udies using this gene in a similar manner [31,32], Ppia showed no sign of circadian or ultradian variation (data not shown). The comparative CT method was similarly applied to determine the relative expression of Bmal1 mRNA. The following primers were used for real-time PCR analysis: Bmal1 forward: 59- CCAAGAAAGTATGGACACAGACAAA39; Bmal1 reverse: 59- GCATTCTTGATCCTTCCTTGGT-39; Ppia forward: 59- TGTGCCAGGGTGGTGACTT-39; Ppia reverse: 59- TCAAATTTCTCTCCGTAGATGGACTT39.Experiment 2: Mutagenic Analysis of Putative Binding Sites Mediating miR-142-3p-induced Repression of Bmal1 39 UTR ActivitymiTargetTM miRNA Target Sequence 39 UTR Expression Clone containing Bmal1 39 UTR sequence (Accession: NM_007489.3) inserted in the pEZX-MT01 vector was purchased from GeneCopoeia, Inc (Rockville, MD). The plasmid was propagated using methods established in our previous study [26]. Deletions in predicted miR-142-3p binding sites on the Bmal1 16574785 39 UTR were generated using QuikChange II XL SiteDirected Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocols. Briefly, the full-length Bmal1 39 UTR was PCR mutagenized using specific primers so as to delete nucleotides 1? complementary to miR-142-3p seed region. After Dnp1-mediated degradation of parental plasmid DNA, the mutagenized plasmid was transformed into XL10-Gold Ultracompetent cells (Stratagene) and transformants were selected on kanamycin-containing (final conc. = 50 mg/ml) imMedia agar plates (Invitrogen). A single colony was isolated and propagated in imMedia Kan+ liquid medium (final conc. = 50 mg/ml). TheBmal1 constructs.UTRluciferasereportermiR-142-3p Modulation of BMAL1 in SCN Pacemakerplasmid was extracted using HiSpeed Plasmid Midi Kit (Qiagen, Inc.) and then sequenced to verify the targeted deletion (Bmal1 c.1_7del). Identical methods were also used to delete nucleotides 335?57 corresponding to a second predicted miR-142-3p binding site on the Bmal1 39 UTR complementary to the seed region along with additional nucleotides that may function as a 39 supplementary or compensatory element and aid in miRNA biological activity [33?5]. The resulting plasmid (Bmal1 c.335_357del) was then subjected to a second round of mutagenesis to provide for additional deletion of nucleotides 1?. The plasmid with targeted deletions of both miR-142-3p target sites on the Bmal1 39 UTR (Bmal1 c.1_7del+c.335_357del) was propagated and sequenced to verify these deletions as described above. The miRNA 39 UTR target control vector (Genecopoeia; CmiT000001-MT01), consisting of the pEZX-MT01 vector backbone without any 39 UTR sequence, was used to determine the specificity of miRNA interactions with the full-length and mutagenized Bmal1 vectors. miR-142-3p-mediated regulation of Bmal1 39 UTR was analyzed in human embryonic kidney (HEK293) cells using established methods [26]. Briefly, cells were seeded onto 24-well plates (Corning, Inc., Tewksbury MA) and 24 h later were co-transfected with pEZX-MR04 miR-142 expression vector (miExpress Precursor miRNA expression clone; Genecopoeia, Inc., MmiR3437MR04) and with either the target control, full-length Bmal1 39 UTR (WT), Bmal1 23977191 c.1_7del, Bmal1 c.335_357del or Bmal1 c.1_7del+c.335_357del miRNA 39 UTR target clones. As an additional control for specificity of the deletion, cells were also co-transfected with miR-494 expression vector and either the target control, Bmal1 or Bmal1 c.1_7del+c.335_357del miRNA 39 UTR target clones. Following transfection for.
