Journal: Science signaling
Article Title: Identification of the miR-106b~25 MicroRNA Cluster as a Proto-Oncogenic PTEN-Targeting Intron That Cooperates with Its Host Gene MCM7 in Transformation
Figure Lengend Snippet: Pb/MCM7i13 transgenic construct. (A) Pb, prostate-specific rat probasin promoter ARR 2 PB; black box, HA tag; white box, Xpress tag; gray boxes, human MCM7 coding sequence; red and blue boxes, miR-93/106b and miR-25 genes; yellow box, SV40 poly(A) (SV40 pA); orange box, probe used for Southern analysis of transgenic lines; green arrows, primers used for PCR analysis of transgenic lines and for genotyping. (B) Top: Southern blot analysis. Genomic DNA extracted from tails was digested with Bam HI and hybridized with a probe complementary to the SV40 poly(A) region as shown in (A). The Bam HI–digested Pb/MCM7i13 plasmid was used as positive control (C+). Bottom: PCR analysis. A forward primer complementary to the probasin promoter and a reverse primer complementary to the tag were used, as shown in (A). Empty Pb and Pb/MCM7i13 plasmids were used as negative (C−) and positive (C+) controls. L, 100-bp ladder. Three representative positive (P) and negative (N) mice are shown. (C) Abundance of the transgenic construct measured by real-time PCR in nine different lines. A primer pair specific for human MCM7 coding sequence was used. LE, ME, HE: low, medium, and high expressors, respectively. Human MCM7 .
Article Snippet: The following antibodies and reagents were used: antibodies against Hsp-90 #61041 (Becton Dickinson); PTEN #9559 (WB), pAkt (Ser473 ) #9271 (WB), #3787 (IHC), Akt #9272, pS6 #2211 (Cell Signaling); DICER #CG006 (Clonegene); MSCV-neo (Clontech); siGENOME non-targeting small interfering RNA (siRNA) #2 (si-Luc), si-miR-19a, si-miR-19b, si-miR-22, si-miR-25, si-miR-92a, si-miR-17, si-miR-20a, si-miR-93, si-miR-106b, si-miR-302a, si-miR-372, si-miR-373, si-PTEN, siGLO RISC-free control siRNA miRNA antisense inhibitor negative control #1, miR-19a antisense inhibitor, miR-22 antisense inhibitor, miR-25 antisense inhibitor, miR-93 antisense inhibitor, miR-106b antisense inhibitor, Dharmafect 1 (Dharmacon); miR-17, miR-19a, miR-20a, miR-22, miR-25, miR-93, and miR-106b 3′ digoxigenin (DIG)–labeled LNA ISH probes (Exiqon); lipofectamine 2000, Trizol reagent, deoxyribonuclease I (DNase I) amplification grade, SuperScript II reverse transcriptase, Dulbecco's modified Eagle's medium (DMEM), RPMI 1640, fetal bovine serum (FBS), keratinocyte serum-free medium, epidermal growth factor (EGF), bovine pituitary extract (BPE), antibody against Xpress Tag, pcDNA3.1/His C, geneticin (G418), hygromycin, antibody against PTEN (IHC) #51-2400 (Invitrogen); human MCM7 complementary DNA (cDNA) #MHS1011-75176 (Open Biosystems); pGL3-Control ( Firefly luciferase ), pRL-TK ( Renilla luciferase ), Dual-Luciferase reporter assay (Promega); fraction V bovine serum albumin (BSA), polybrene, insulin, antibody against actin #A3853, anti-FLAG antibody #F3165, puromycin (Sigma); QuantiTect Sybr Green PCRkit (Qiagen); antibody against MCM7 #9966 (Santa Cruz Biotechnology); QuikChange II XL Site-Directed Mutagenesis Kit, Herculase Taq polymerase (Stratagene); Ki67 antibody #VP-K451 (Vector Laboratories).
Techniques: Transgenic Assay, Construct, Sequencing, Polymerase Chain Reaction, Southern Blot, Plasmid Preparation, Positive Control, Mouse Assay, Real-time Polymerase Chain Reaction