124 Nucleic Acids Research, 2009, Vol. 37, No. 1 GGC CCC AGT ATG GAA AA. The PCR products were cloned into PCR2.1 plasmids using a TOPO cloning kit (Invitrogen) and the plasmid sequenced. The promoter sequences were ﬁnally subcloned into PGL2 Luciferase reporter plasmids (Promega). RNA isolation and RT-PCR Whole embryos were collected from time mated C57Bl6 mice at the embryonic stages (E) 10.5, 11.5, 12.5, 14.5 and 16.5 (plug day was E0.5, B&K, UK). Total RNA and miRNA was extracted from embryos and N2A cells using RNeasy mini (Qiagen) or miRNA extraction kit (Ambion), respectively. RT-PCR for the analysis of gene expression was performed on 500 ng of RNA and cDNA synthesis was carried out using SuperscriptIII (Invitrogen). RT-PCR analysis of the DNM3, Dynamin- 3 opposite strand (DNM3os) and GAPDH gene expres- sion was performed using previously described primer sequences (6). Optimized HIF-1 and Twist-1 primers were designed using the vector NTI program (Invitrogen). The primer sequences for RT-PCR were: DNM3, (F) CCG AGA CTT AAT TCC AAA ACA ATA ATG; DNM3 (R), AAA GGA GTC ACT GCT GTT GGG AAA A; DNM3os, (F) GGT CTC ACC CTG CTT GTT AAT CAA GGG GGA; DNM3os, (R) TCC TGT TGT TAC TGG CCC TCA TGC AGC C; GAPDH (F), ACC ACA GTC CAT GCC ATC TC; GAPDH (R), TCC ACC ACC CTG TTG CTG TA; HIF-1 (F), TGA GCG TTT CCT AAT CTC AT; HIF-1 (R), AGC GAC AAA GTG CAT AAA AC; Twist-1 (F), CGA CGA CAG CCT GAG CAA CA; Twist-1 (R), TGC AGC TCC TCG TAC GAC TG. PCR was carried out at 948C for 45 s, 608C for 45 s and 728C for 1 min for 25 cycles, using high-ﬁdelity Taq polymerase (Roche). Parallel ampliﬁcations were performed on the templates from which the reverse transcriptase was omitted to ensure that ampliﬁcation was dependent on the presence of single strand of cDNA. Western blot All primary antibodies were diluted in PBS with 5% (v/v) Blocking solution (Roche). The mouse anti-Twist1 (AbCam) Ab was used at a 1:1000 dilution and the mouse anti-a-Tubulin (Sigma) at 1:1000. In all cases, cor- responding horseradish peroxidase-linked antibodies were used (Amersham). RNA isolation and miRNA detection Small RNAs were isolated using the mirVana miRNA Isolation Kit (Ambion). miRNA expression levels were assessed using Taqman MiRNA Assays (Applied Biosystems) according to the manufacturer’s protocol. Results were normalized against levels of RNU6B (as assessed by the corresponding Taqman Assay; Applied Biosystems); 10 ng of total RNA were used as the template for the RT reaction in each case. Luciferase reporter assay Cells (at 50% conﬂuence) in 24-well plates were trans- fected using lipofectamine (Invitrogen) mixed with a Fireﬂy luciferase reporter gene construct (200 ng). Cell extracts were prepared 48 h after transfection and the luci- ferase activity was measured using the Luciferase Reporter Assay System (Promega). Whole-mount in situ hybridization DIG (digioxigenin) labeled DNM3os antisense RNA probes were generated from RT-PCR derived templates by in vitro transcription. In order to produce a DNA tem- plate for the generation of antisense RNA probes by in vitro transcription, T7 RNA polymerase promoter sequences were added to the 50end of the PCR product. The primer sequences used are shown below (promoter sequences are in bold): DNM3os (F), GGT CTC ACC CTG CTT GTT AAT CAA GGG GGA, DNM3os (R): GCA GTA ATA CGA CTC ACT ATA GGG TCC TGT TGT TAC TGG CCC T CA TGC AGC C. One hundred nanograms of template DNA was used for in vitro tran- scription using a DIG-labeling kit (Roche). Pre DIG labeled LNA miR-199a-5p, miR-199a-3p, mir-214 and scrambled control probes were purchased from Exiqon. In situ hybridization with DIG probes and detection with alkline-phosphatase-conjugated antibodies were car- ried out as described previously (17). RESULTS Twist-1 drives the expression of miR-199a/214 The miR-199a/214 gene cluster is located in the opposite (30–50) direction to the DNM-3 gene suggesting it is tran- scribed from its own promoter (Figure 1). Furthermore, it was predicted previously that the expression of a (non- coding) 7.9-kb Dynamin-3 opposite strand (DNM3os) during development may be under the control of Twist- 1(6). Hence, we investigated whether Twist-1 drives the expression of DNM3os, miR-199a and miR-214 Figure 1. The miR-199a/214 cluster lies within the DNM3 gene. A schematic diagram of the Dynamin3 gene showing the complementary strand within its intron encodes a single transcript (DNM3os). The inset ﬁgure shows the DNM3os transcript encodes miR-199a and miR-214 and that an E-Box promoter drives transcription in a reverse orientation.