Journal: EBioMedicine

Article Title: A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies

doi: 10.1016/j.ebiom.2014.10.013

Figure Lengend Snippet: Effect of CD22ΔE12 vs. CD22 WT on clonogenicity and self-renewal rate of ALL cells. [A1] Depicted is a representative 1% agarose gel showing the PCR products of FL CD22 and CD22ΔE12–14. [A.2] Subcloning of the CD22 FL or CD22ΔE12–14 genes into the lentiviral pCL6-2AEGwo vector through NheI and XhoI restriction sites was confirmed through restriction enzyme digestion and DNA sequencing. Depicted is a representative 1% agarose gel showing that the generated lentiviral constructs have the correct size FL CD22 (2.5-kb) or CD22ΔE12–14 (2.2-kb) inserts. [B.1] In order to confirm that the lentiviral vectors can be used to achieve expression of FL and truncated CD22 in human cells, 293T cells were transfected with lentiviral constructs for FL CD22, CD22ΔE12–14, as well as pCL6-2AEGwo lentiviral vector without any subcloned CD22. 48 h post transfection cells were examined for FL CD22 and CD22ΔE12–14 mRNA by RT-PCR using the P7, P9, and P10 primer pairs. The P7 primer set was used to amplify a 182-bp region (c.2180–c.2361) of the CD22 cDNA extending from Exon 11 to Exon 13 and spanning the entire Exon 12. The P9 primer set was used amplify a 160-bp region (c.2304–c.2463) of the CD22 cDNA extending from Exon 12 to Exon 14 and spanning the entire Exon 13. The P10 primer set was used to amplify a 213-bp region (c.433–c.645) of Exon 4 of the CD22 cDNA present in both wildtype CD22 and CD22ΔE12–14 mRNA species. As expected all primer sets yielded PCR products in cells transfected with the FL CD22 vector and only the P10 primer set yielded a PCR product in cells transfected with the CD22ΔE12–14 vector. [B.2 and B3] The increased expression levels of the full-length and truncated proteins in transduced 293-T cells (depicted in B.2) and ALL-1 cells (depicted in B.3) were confirmed to be similar by Western blot analysis done at 96 h post-transduction. [C D] ALL cell lines DAUDI (Burkitt's leukemia/B-ALL) (panels C1 C.2) and ALL1 (BCR-ABL + B-precursor ALL) (panel C3) were transduced (trans) with wildtype and mutant human CD22 genes using the pCL6-2AEGwo lentiviral vector and then assayed for colony formation in semi-solid methylcellulose cultures without additional stroma support or cytokines. Colony formation was examined using an inverted Nikon Eclipse TS100 microscope with an Epifluorescence attachment. Images were taken using a Digital Sight DS-2MBW Nikon camera (System magnification: 100× or 200 × as indicated). Green fluorescence of colonies resulting from GFP expression confirms successful transduction of the cell lines. [D] Depicted are bar graphs comparing the mean colony numbers in cultures of untransduced and transduced cells.

Article Snippet: The correct PCR products (FL: 2541-bp and ΔE12–14: 2208-bp) were ligated into the 8497-bp lentiviral vector pCL6-2AEGwo through the NheI and XhoI restriction sites (underlined) using the Quick Ligase kit (New England Biolabs catalog no. M2200L) following the manufacturer's instructions.

Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Subcloning, Plasmid Preparation, DNA Sequencing, Generated, Construct, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transduction, Mutagenesis, Microscopy, Fluorescence

Journal: Brazilian Journal of Microbiology

Article Title: Expression of mouse beta defensin 2 in escherichia coli and its broad-spectrum antimicrobial activity

doi: 10.1590/S1517-838220110003000043

Figure Lengend Snippet: Schematic representation of the expression vector, pET32a-mBD2. The cDNA for mature mBD2 were amplified using PCR from the pcDNA3.1(+)-mBD2 plasmid. PCR product was cleaved by Xho I and Kpn I, and the mBD2 fragment was inserted into similarly digested pET32a(+) vector to construct the expression vector, pET32a-mBD2.

Article Snippet: The PCR conditions were following: 94°C, 4 min; 30 cycles of 94°C, 30 s; 58°C, 30 s; 72°C, 30 s; 72°C, 5 min. PCR product was cleaved by Xho I and Kpn I , and the mBD2 fragment was inserted into similarly digested pET32a(+) (Novagen, Shanghai, China) to construct the expression vector pET32a-mBD2.

Techniques: Expressing, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Construct

Journal: Osong Public Health and Research Perspectives

Article Title: Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei

doi: 10.1016/j.phrp.2015.10.003

Figure Lengend Snippet: (A) Digestion of pTY-α-amylase with Nhe I and Xho I restriction enzymes. Lane 1: pTYB2 digested with Nhe I and Xho I; lane2: pTY-α-amylase digested with Nhe I and Xho I; lane 3: PCR product of α-amylase. (B) SDS-PAGE analysis of α-amylase expression. Lane 1: before IPTG induction; lane 2: after IPTG induction; lane 3: 16 hours after IPTG induction; lane 4: 20 hours after IPTG induction. (C) Western blot analysis of α-amylase with anti-intein polyclonal antibody. Lane1: α-amylase; lane 2: lysate of untransformed bacteria. IPTG = isopropyl- β-d-thiogalactopyranoside; M = molecular marker; SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Article Snippet: P. woesei chromosomal DNA was used as a source of the α-amylase gene and polymerase chain reaction (PCR) amplification was done with the following primers: 5′- GCTAGC TTGGAGCTTGAAGAGGGAG-3′ and 5′- GAGACC ACAATAACTCCATACGGAG-3′ containing recognition sites for restriction endonucleases Nhe I and Xho I (Fermentas; Vilnius, Lithuania).

Techniques: Polymerase Chain Reaction, SDS Page, Expressing, Western Blot, Marker, Polyacrylamide Gel Electrophoresis

Journal: BioMed Research International

Article Title: Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable β-Glucosidase from Clostridium thermocellum in Escherichia coli

doi: 10.1155/2019/9308593

Figure Lengend Snippet: Graphical representation of pET28a-bglA. Expression vector was created by insertion of bglA gene between NcoI and XhoI sites. Inserted sequence was adjacent to lac operator and terminates at 6xHis-tag included in the frame. Kan R : kanamycin resistance gene, Ori: origin of replication. The sketch was created using SnapGene® software (GSL Biotech).

Article Snippet: NcoI and XhoI (Fermentas Inc., Ontario, Canada) restriction enzymes were used for restriction of cloning and expression vectors.

Techniques: Expressing, Plasmid Preparation, Sequencing, Software

Journal: Protein expression and purification

Article Title: Expression of Recombinant Human Mast Cell Chymase with Asn-linked Glycans in Glycoengineered Pichia pastoris

doi: 10.1016/j.pep.2014.08.005

Figure Lengend Snippet: pJ915-rhChymase vector map The P. pastoris ] using 5’ XhoI and 3’ NotI restriction sites. The GAP promoter provides constitutive expression of the fusion protein and the plasmid contains a Zeocin resistance selectable marker gene. The plasmid is linearized at the SwaI restriction site prior to electroporation.

Article Snippet: The DNA encoding human Chymase (hChymase) with an added N-terminal Kex2 protease cleavage site was isolated from the previously described plasmid pPICzα-rhChymase [ ] using simultaneous digestion with XhoI and NotI restriction endonucleases (Thermo Scientific, FastDigest).

Techniques: Plasmid Preparation, Expressing, Marker, Electroporation

Journal: Applied and Environmental Microbiology

Article Title: Isolation and Characterization of a Cyanophage Infecting the Toxic Cyanobacterium Microcystis aeruginosa

doi: 10.1128/AEM.72.2.1239-1247.2006

Figure Lengend Snippet: Ma-LMM01 genomic DNA digested with various restriction enzymes. Lanes 1 and 18, 1-kb ladder marker; lane 2, BamHI; lane 3, EcoRI; lane 4, EcoRV; lane 5, HincII; lane 6, HindIII; lane 7, NotI; lane 8, PstI; lane 9, SacI; lane 10, SalI; lane 11, ScaI; lane 12, SmaI; lanes 13 and 14, lambda/HindIII marker; lane 15, SpeI; lane 16, XhoI; lane 17, XbaI.

Article Snippet: Using the manufacturers' recommendations, we tested the sensitivity of the phage nucleic acid to RNase A (0.01 ng μl−1 ; 37°C for 1 h; Nippon Gene Co., Ltd.), DNase I (0.02 ng μl−1 ; 37°C for 1 h; Promega Co., Ltd.), and the following 14 restriction enzymes, which were incubated for 16 h: SpeI (0.5 U μl−1 ; 37°C; TOYOBO Co., Ltd.), XhoI (0.45 U μl−1 ; 37°C; TOYOBO Co., Ltd.), XbaI (0.5 U μl−1 ; 37°C; Roche Molecular Biochemicals), BamHI (0.6 U μl−1 ; 37°C; TOYOBO Co., Ltd.), EcoRI (0.5 U μl−1 ; 37°C; TOYOBO Co., Ltd.), EcoRV (0.6 U μl−1 ; 37°C; TOYOBO Co., Ltd.), HincII (0.5 U μl−1 ; 37°C; TOYOBO Co., Ltd.), HindIII (0.5 U μl−1 ; 37°C; Nippon Gene Co., Ltd.), NotI (0.5 U μl−1 ; 37°C; New England Biolabs Inc.), PstI (0.5 U μl−1 ; 37°C; TOYOBO Co., Ltd.), SacI (0.5 U μl−1 ; 37°C; TOYOBO Co., Ltd.), SalI (0.5 U μl−1 ; 37°C; TOYOBO Co., Ltd.), ScaI (0.5 U μl−1 ; 37°C; TOYOBO Co., Ltd.), and SmaI (0.6 U μl−1 ; 30°C; TOYOBO Co., Ltd.).

Techniques: Marker