Geneoptimizer

The canine (c) FVIII cDNA^{31 (link)} (Figure 1a) was used to generate Lenti-EF1α(–5.5HSCR)-cFVIII. This construct contains a BDD cFVIII transgene under the control of the ubiquitous EF1α promoter into which an endothelial enhancer element was added.^{30 (link)} MunI sites at amino acids (AAs) 749 and 1,663 were used to remove the B domain while retaining the thrombin cleavage sites at AA 734 and 1681 (BDD cFVIII: Figure 1b). This DNA sequence was used for codon optimization and additionally includes the first 269 amino acids of the B domain that contains six putative glycosylation sites bounded by SQa and SQb^{19 (link)} linker sequences (CO-N6 cFVIII: Figure 1c). The nucleotide sequences directly 5’ to the ATG start of translation were modified to introduce a Kozak consensus sequence and an additional TGA stop codon was added to the 3’ end of the gene. XhoI and XmaI restriction sites were added to the 5’ and 3’ ends of the CO-N6 cFVIII sequence to facilitate cloning into the lentiviral plasmid. The CO-N6 cFVIII cDNA was generated for a canine codon usage bias with GeneOptimizer, a proprietary software program developed by GeneArt and was manufactured by GeneArt (Invitrogen, Carlsbad, CA) and cloned into the Lenti-EF1α(–5.5HSCR)-cFVIII, thereby replacing the BDD cFVIII transgene to generate Lenti-EF1α(–5.5HSCR)-CO-N6-cFVIII.

GePTS1 coding sequence (GenBank™ accession no. LC121822), as well as four individual mutants (D50A, D83A, Y103F, and Y181F) were codon-optimized for E. coli and synthesized via GeneOptimizer^® (Invitrogen) without the N-terminal signal peptide (23 amino acids). The GePTS1 gene was cloned into the pET101/D-TOPO^®E. coli expression vector, whereas the four mutants were cloned into the pET100/D-TOPO^®. GePTS1 and each of the four mutant constructs were transformed into One Shot^® BL21 Star™ (DE3) competent E. coli (Invitrogen) according to the manufacturer's protocol. Initial Luria-Bertani medium cultures (10 ml) containing 100 μg/ml carbenicillin were incubated overnight (∼15 h) at 37 °C with shaking at 250 rpm. A 500-μl aliquot of each culture was then used to inoculate Luria-Bertani medium (50 ml) containing 100 μg/ml carbenicillin. After incubating at 37 °C with shaking at 250 rpm to obtain an A₆₀₀ of ∼0.6, the cultures were induced with isopropyl 1-thio-β-d-galactopyranoside at a final concentration of 1 mm. After continued shaking at 28 °C for 24 h, cells were harvested by centrifugation at 3,000 × g for 20 min at 4 °C, with the pellets frozen and stored at –80 °C.

DNA-sequences coding for papillomavirus capsid proteins L1 and L2 were codon-optimized for expression in human cells using GeneArt’s GeneOptimizer and synthesized by GeneArt (Invitrogen, Regensburg, Germany). Sequences are available in the supporting information S1 File. L1 and L2 sequences were cloned into the multiple cloning site of the mammalian expression vector pcDNA3.1+ (Invitrogen) via HindIII and XhoI. The two sequences were connected by an IRES-sequence (GenBank: X68257.1), which was synthesized by GeneArt and cloned into the vector via AflIII and NheI. Cloning as well as plasmid propagation was performed in E.coli DH5α (New England Biolabs).
Plasmids for transfection were prepared using a NucleoBond Plasmid PC500 Maxiprep Kit (Macherey Nagel).
As reporter plasmids, pCMV-Gluc-1 luciferase of Gaussia princeps (Targeting Systems/NEB)), pCR-Luc3 (firefly luciferase reporter) and pEGFP-C1 (GFP reporter; GenBank U55763.1) were used.