pgem t cdna  (Sino Biological)

Journal: PLoS ONE

Article Title: Hypothalamic-Specific Manipulation of Fto , the Ortholog of the Human Obesity Gene FTO , Affects Food Intake in Rats

doi: 10.1371/journal.pone.0008771

Figure Lengend Snippet: ( A ) Fto expression, quantified by real-time RT-PCR, is up-regulated in the arcuate nucleus following a high-fat diet demonstrated by the relative arcuate Fto mRNA expression in rat either fed on a standard chow or on a high-fat diet for 10 weeks. Data is represented as the mean±S.E.M of at least 6 independent rats per group; **P<0.01. ( B ) Fto mRNA is diffusely expressed throughout the arcuate nucleus and overlaps with Pomc neurons. Double in situ hybridization detecting Fto ( 35 S labeled) and Pomc (DIG labeled) mRNA in the cells of the arcuate nucleus. Insert in figure shows a high-magnification (scale bar, 20 µm) of a Pomc containing neurons colocalised with Fto (marked with *). ( C ) Intra-nuclei bilateral injections of adeno-associated virus (AAV2/7) mediated transfer of GFP cDNA precisely targets the hypothalamic arcuate (ARC) and paraventricular (PVN) nuclei; as demonstrated by photomicrographs of representative coronal sections showing localization of GFP 7 days after injection; right panels, higher magnification of area indicated by red box. Scale bar, 100µm. 3v: third ventricle; ME: median eminence.

Article Snippet: Dig-labelled probes for sense and antisense Pomc mRNA were generated from the cDNA template corresponding to exon3 of Pomc in a pGem- T vector (Promega) and transcribed by SP6 or T7 RNA polymerase (Progema).

Techniques: Expressing, Quantitative RT-PCR, In Situ Hybridization, Labeling, Injection

Journal: BMC Research Notes

Article Title: Three non-autonomous signals collaborate for nuclear targeting of CrMYC2, a Catharanthus roseus bHLH transcription factor

doi: 10.1186/1756-0500-3-301

Figure Lengend Snippet: Schematic representation of the full-length CrMYC2 protein showing the positions of the potential nuclear localization signal sequences (NLS 1, 2, 3, and 4) and of the regions of CrMYC2 used to make the GFP fusions . NLS: nuclear localization signal, CrMYC2: full-length CrMYC2 sequence, F1 to F8: partial CrMYC2 sequences used for fusion to GFP. Grey box: bHLH domain. Single oval shape: monopartite NLS. Double oval shape: bipartite NLS.

Article Snippet: PCR cycling conditions were as follows: 94°C for 4 min (1 cycle) followed by 94°C for 1 min, an annealing step at various temperatures depending on the T m of the primers used for 1.5 min, and 72°C for 1 min (30 cycles), with a final extension step at 72°C for 5 min. PCRs were performed in a final volume of 25 μl with 0.25 U of Taq polymerase and 1 × MgCl 2 -free buffer (Promega), 2 mM MgCl 2 , 200 nM of each dNTP, appropriate oligonucleotides (1 μM each) and 5 ng of Crmyc2 cDNA in pGEM-T Easy (Promega).

Techniques: Sequencing

Journal: BMC Research Notes

Article Title: Three non-autonomous signals collaborate for nuclear targeting of CrMYC2, a Catharanthus roseus bHLH transcription factor

doi: 10.1186/1756-0500-3-301

Figure Lengend Snippet: Alignment of amino acid sequences of CrMYC2, AtMYC2/RAP1/AtbHLH006/At1g32640.1, At4g17880.1, At5g46760.1, At5g46830.1 and Os10g42430.1 . Bold letters correspond to conserved amino acids residues within a putative NLS. Basic amino acids are boxed in grey. Asterisks under sequences correspond to positions where the amino acid is the same for all sequences. Boxed or underlined sequences indicate the three regions involved in CrMYC2 nuclear targeting.

Article Snippet: PCR cycling conditions were as follows: 94°C for 4 min (1 cycle) followed by 94°C for 1 min, an annealing step at various temperatures depending on the T m of the primers used for 1.5 min, and 72°C for 1 min (30 cycles), with a final extension step at 72°C for 5 min. PCRs were performed in a final volume of 25 μl with 0.25 U of Taq polymerase and 1 × MgCl 2 -free buffer (Promega), 2 mM MgCl 2 , 200 nM of each dNTP, appropriate oligonucleotides (1 μM each) and 5 ng of Crmyc2 cDNA in pGEM-T Easy (Promega).

Techniques:

Journal: BMC Research Notes

Article Title: Three non-autonomous signals collaborate for nuclear targeting of CrMYC2, a Catharanthus roseus bHLH transcription factor

doi: 10.1186/1756-0500-3-301

Figure Lengend Snippet: Subcellular localization in epidermal onion cells of fusions of GFP with full-length CrMYC2, with different putative nuclear localization signals and with deletion mutants of CrMYC2 . The green fluorescence of the GFP protein was visualized by epifluorescence microscopy. A: control with GFP alone. B: control with GFP fused to the NLS of the SV40 large T antigen. C: control with GFP fused to an endoplasmic reticulum retention signal. D: right, GFP fused to full-length CrMYC2, left: corresponding DAPI staining,. E-H: GFP fused with different putative NLS sequences from CrMYC2, 1, 2, 3 and 4, respectively; I-P: GFP fused with different deletion mutants of CrMYC2 (F1 to F8).

Article Snippet: PCR cycling conditions were as follows: 94°C for 4 min (1 cycle) followed by 94°C for 1 min, an annealing step at various temperatures depending on the T m of the primers used for 1.5 min, and 72°C for 1 min (30 cycles), with a final extension step at 72°C for 5 min. PCRs were performed in a final volume of 25 μl with 0.25 U of Taq polymerase and 1 × MgCl 2 -free buffer (Promega), 2 mM MgCl 2 , 200 nM of each dNTP, appropriate oligonucleotides (1 μM each) and 5 ng of Crmyc2 cDNA in pGEM-T Easy (Promega).

Techniques: Fluorescence, Epifluorescence Microscopy, Staining

Journal: BMC Research Notes

Article Title: Three non-autonomous signals collaborate for nuclear targeting of CrMYC2, a Catharanthus roseus bHLH transcription factor

doi: 10.1186/1756-0500-3-301

Figure Lengend Snippet: Oligonucleotides used for the amplification of full-length or deletion mutants of Crmyc2 or for the reconstitution of sequences encoding the putative NLSs of the CrMYC2 protein

Article Snippet: PCR cycling conditions were as follows: 94°C for 4 min (1 cycle) followed by 94°C for 1 min, an annealing step at various temperatures depending on the T m of the primers used for 1.5 min, and 72°C for 1 min (30 cycles), with a final extension step at 72°C for 5 min. PCRs were performed in a final volume of 25 μl with 0.25 U of Taq polymerase and 1 × MgCl 2 -free buffer (Promega), 2 mM MgCl 2 , 200 nM of each dNTP, appropriate oligonucleotides (1 μM each) and 5 ng of Crmyc2 cDNA in pGEM-T Easy (Promega).

Techniques: Amplification