Journal: Nucleic Acids Research

Article Title: Targeted isolation of cloned genomic regions by recombineering for haplotype phasing and isogenic targeting

doi: 10.1093/nar/gkr668

Figure Lengend Snippet: Fosmid library host optimization. ( A ) Recombineering assay with GB05RedTrfA + pSC101β. The strain carries in the genome the modified red operon ( gam , beta , exo , recA ) (red) under the control of the rhamnose inducible Rha promoter and has the TrfA gene (blue), which promotes high copy fosmid replication under the control of the arabinose inducible BAD promoter. The BAD promoter also drives expression of the Redβ protein (red) located on the helper plasmid pSC101β. A random fosmid clone was chosen for the insertion of modified blasticidin (bsd) cassette via recombineering. The cassette is flanked with 50 bp homology arms, identical to the region of choice (green). After the selection step the temperature-sensitive plasmid pSC101β is lost at 37°C. OriV—bidirectional origin of replication, Fori—unidirectional origin of replication. ( B ) Strain history. All strains were derived from DH10B. The strains GB05, GB05Red and DY380 as well as the pSC101γβαA plasmid were described previously . The BADTrfA and RhaγβαA cassettes in GB05RedTrfA were inserted at ybcC locus as the BADγβαA cassette in GB05Red. ( C ) Comparison of the recombineering efficiencies of the host strains. To test recombineering efficiency, a modified blasticidin cassette was inserted in a randomly selected fosmid from the H7 library. The number of recombinants was normalized to the number of cells surviving electroporation. Higher recombineering frequencies were obtained using the pSC101β helper plasmid. High copy fosmid induction further promoted recombineering efficiency.

Article Snippet: The P BAD TrfA was amplified from the genome of E. coli EPI300 (Epicentre Biotechnologies, Madison, WI, USA) and added by recombineering to the P Rha redγβαrecA.

Techniques: Modification, Expressing, Plasmid Preparation, Selection, Derivative Assay, Electroporation

Journal: BMC Cancer

Article Title: Methylthioadenosine (MTA) inhibits melanoma cell proliferation and in vivo tumor growth

doi: 10.1186/1471-2407-10-265

Figure Lengend Snippet: Methylthioadenosine (MTA) promotes cytostatic effects rather than pro-apoptotic effects in melanoma tumors and cells . (A) Tumor sections from mice treated either with DMSO or MTA were stained with cyclin D1 and Ki67 antibodies. Representative pictures are shown. (B) Quantification of apoptotic cells within the tumors. Paraffin-embedded tumor samples were subjected to TUNEL assay or stained against cleaved caspase-3. Graph shows the quantification of the TUNEL assay. Positive cells from ten fields (20×) per sample were quantified and the average number of cells per field was calculated. (C) Upper graph, quantification by qRT-PCR of VEGF levels in tumor samples. Lower graph, qRT-PCR of VEGF expression levels. 37-31E were untreated (Control) or treated for 48 h with 10 μM of MTA in complete medium. Microvessel's density quantification in xenografts. Graph shows CD31 fluorescence per μm 2 . Representative pictures are showed on the right. p -values were calculated performing a t -student test. (D) 37-31E cells were treated with MTA (10 μM) for the time points indicated. Fifty micrograms of total lysates were resolved by PAGE-SDS. p-Bad, cleaved-caspase3, p-S6 and cyclin D1 protein levels are showed. GAPDH is used as a loading control. (E) MTA treatment induces a slowdown cell cycle G1 phase. Cells were grown in complete medium for 48 h in the presence or absence of MTA (10 μM). Cell cycle analysis was measured in triplicates using Cell Cycle Analysis Guava-Viacount reagent (Guava Technologies). Average of the three samples in each phase of the cell cycle are shown. p -values were calculated performing a t -student test (* = p < 0.05; ** = p < 0.01).

Article Snippet: Cyclin D1 antibody was from Santa Cruz; phospho-Erk1/2 (Thr202/Tyr204), cleaved-caspase-3, p-S6 (Ser235/236), and phospho-Akt (Thr308) antibodies were from Cell Signaling (Danvers, MA USA); p-Bad was from Genscript, Piscataway, NJ, USA, Ki67 was from Master diagnostica, (Granada, Spain) and GAPDH was from Trevigen (Gaitherburg, MD, USA).

Techniques: Staining, TUNEL Assay, Quantitative RT-PCR, Expressing, Control, Fluorescence, Cell Cycle Assay