codon optimization and dna synthesis for all three constructs  (Gene Universal Inc)

Journal: Frontiers in Pharmacology

Article Title: Aptamer-Driven Toxin Gene Delivery in U87 Model Glioblastoma Cells

doi: 10.3389/fphar.2021.588306

Figure Lengend Snippet: Schematic representation of APTSAP, APTSAPKQ and pBlue_SAP DNA sequences. The nucleotide sequence of AS1411 [5′-d (GGT​GGT​GGT​GGT​TGT​GGT​GGT​GGT​GG)-3] was placed at the 5′ terminus of a synthetic construct containing the CMV promoter, the saporin gene, a chimeric intron derived from pCI vector, and a poly-adenylation signal at the 3′ terminus and inserted into pBluescript II KS (+) vector using a SmaI restriction site. In the APTSAPKQ construct the saporin gene sequence was substituted with the saporin KQ mutated sequence. The pBlue_SAP construct lacks the 5′ AS1411 aptamer sequence. In the scheme we also indicated the PCR primers at their relative positions (FW1/RV1, FW2/RV2, FW3/RV3). This map was created by the SnapGene Viewer freeware software (“SnapGene software” from Insightful Science; available at http://snapgene.com ).

Article Snippet: APTSAP, APTSAPKQ and pBlue_SAP vectors DNA synthesis and codon optimization for mammalian cell expression were from GenScript (United States), while the single strands corresponding to AS1411 (ssAPT) was purchased from Eurofins Genomics (Germany).

Techniques: Sequencing, Construct, Derivative Assay, Plasmid Preparation, Software

Journal: Frontiers in Pharmacology

Article Title: Aptamer-Driven Toxin Gene Delivery in U87 Model Glioblastoma Cells

doi: 10.3389/fphar.2021.588306

Figure Lengend Snippet: Dose-response curves showing respectively the effects of ssAPT, APTSAP, APTSAPKQ, pBlue_SAP and pBluescript II KS (+) on U87MG cells. Cells (1.6 × 10 3 /well) were seeded in 96-well plates in a final volume of 200 µl of medium (5% FBS) containing the appropriate concentrations (0, 0.001, 0.01, 1, 10, 50 μmol/L) of ssAPT or the appropriate concentrations (0, 0.001, 0.01, 0.02, 0.05 and 0.1 μmol/L) of APTSAP, APTSAPKQ, pBlue_SAP and pBluescript II KS (+) for 96 h. All the plasmids were denatured at 95°C for 2 min in 10 mM Tris/HCl buffer pH 7.5 containing 100 mM NaCl and 2.5 mM KCl immediately before use. Cell viability was examined using a MTS assay and expressed as percentage of control untreated cells. ssAPT IC 50 : 4.30 × 10 −6 ± 1.70 × 10 −6 M (mean ± S.E.M.); APTSAP IC 50 : 1.30 × 10 −8 ± 2.42×10 −9 M (mean ± S.E.M.). The data reported are representative of a set of at least three independent experiments and are reported as mean ± SD.

Article Snippet: APTSAP, APTSAPKQ and pBlue_SAP vectors DNA synthesis and codon optimization for mammalian cell expression were from GenScript (United States), while the single strands corresponding to AS1411 (ssAPT) was purchased from Eurofins Genomics (Germany).

Techniques: MTS Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Aptamer-Driven Toxin Gene Delivery in U87 Model Glioblastoma Cells

doi: 10.3389/fphar.2021.588306

Figure Lengend Snippet: Representative images observed with an optical microscope of U87 cells after 96h of treatment. Controls (A) ; ssAPT 25 µM (B) ; APTSAP 20 nM (C) ; APTSAPKQ 20 nM (D) ; pBlue_SAP 20 nM (E) .

Article Snippet: APTSAP, APTSAPKQ and pBlue_SAP vectors DNA synthesis and codon optimization for mammalian cell expression were from GenScript (United States), while the single strands corresponding to AS1411 (ssAPT) was purchased from Eurofins Genomics (Germany).

Techniques: Microscopy

Journal: Frontiers in Pharmacology

Article Title: Aptamer-Driven Toxin Gene Delivery in U87 Model Glioblastoma Cells

doi: 10.3389/fphar.2021.588306

Figure Lengend Snippet: Dose-response curves showing respectively the effect of ssAPT, APTSAP, APTSAPKQ, pBlue_SAP and pBluescript II KS (+) on control NIH3T3 cells. Cells (1.6 × 10 3 /well) were seeded in 96-well plates in a final volume of 200 ul of medium (5% FBS) containing appropriate concentrations (0, 0.001, 0.01, 1, 10, 50 μmol/L) of ssAPT and appropriate concentrations (0, 0.001, 0.01, 0.02, 0.05 and 0.1 μmol/L) of APTSAP, APTSAPKQ, pBlue_SAP and pBluescript II KS (+) and for 96 h. All the plasmids were denatured at 95°C for 2 min in 10 mM Tris/HCl buffer pH 7.5 containing 100 mM NaCl and 2.5 mM KCl immediately before use. Cell viability was examined using a MTS assay and expressed as percentage of control untreated cell. The data presented are representative of a set of at least three independent experiments and are reported as mean ± SD.

Article Snippet: APTSAP, APTSAPKQ and pBlue_SAP vectors DNA synthesis and codon optimization for mammalian cell expression were from GenScript (United States), while the single strands corresponding to AS1411 (ssAPT) was purchased from Eurofins Genomics (Germany).

Techniques: Control, MTS Assay

Journal: Frontiers in Pharmacology

Article Title: Aptamer-Driven Toxin Gene Delivery in U87 Model Glioblastoma Cells

doi: 10.3389/fphar.2021.588306

Figure Lengend Snippet: Representative images observed with an optical microscope of NIH3T3 cells after 96 h of treatment. Controls (A) ; ssAPT 25 µM (B) ; APTSAP 20 nM (C) ; APTSAPKQ 20 nM (D) ; pBlue_SAP 20 nM (E) .

Article Snippet: APTSAP, APTSAPKQ and pBlue_SAP vectors DNA synthesis and codon optimization for mammalian cell expression were from GenScript (United States), while the single strands corresponding to AS1411 (ssAPT) was purchased from Eurofins Genomics (Germany).

Techniques: Microscopy

Journal: Frontiers in Pharmacology

Article Title: Aptamer-Driven Toxin Gene Delivery in U87 Model Glioblastoma Cells

doi: 10.3389/fphar.2021.588306

Figure Lengend Snippet: U87 (A) and 3T3 cells (B) were treated with 20 nM APTSAP for 96 h, total RNA was extracted from cell pellets. PCR was performed on the corresponding cDNA using the three couples of primers, as described in the Materials and Methods section. pET11d plasmid carrying the wild type saporin gene (pET11d-SAP) was used as a positive control. Amplified products are indicated by arrows, and the expected dimensions are: FW1/RV1:765 bp, FW2/RV2: 204 bp, FW3/RV3: 621bp. Lanes 1-3-5: pET11d-SAP; lanes 2-4-6: treated.

Article Snippet: APTSAP, APTSAPKQ and pBlue_SAP vectors DNA synthesis and codon optimization for mammalian cell expression were from GenScript (United States), while the single strands corresponding to AS1411 (ssAPT) was purchased from Eurofins Genomics (Germany).

Techniques: Plasmid Preparation, Positive Control, Amplification