Cell specific productivity of HD CAVGFP and corresponding contamination with helper JBΔ5.

<p>Errors correspond to 25% inter-assay variability. The contamination levels of JBΔ5 correspond to the ratio between the I.P titers of JBΔ5 and HD CAVGFP.</p

New Low-Toxicity Cholinium-Based Ionic Liquids with Perfluoroalkanoate Anions for Aqueous Biphasic System Implementation

This work explores the widening of properties of cholinium-based ionic liquids (ILs) through their combination with perfluoroalkanoate anions so that higher number of aqueous biphasic systems (ABSs) containing nontoxic cholinium-based ILs is available. For that purpose, six cholinium perfluoroalkanoate ILs were synthesized and their cytotoxicity was evaluated using three different animal cell lines, envisaging biotechnology applications. Ternary phase equilibrium data for ABSs composed of the cholinium perfluoroalkanoate, with fluoroalkyl chains from C₂ up to C₇, using a strong salting out agent, K₃PO₄, were determined at 25 °C. The results show the relevant role of the size of fluorinated alkyl chain length in the anion since, contrary to other ABSs containing ILs with increasing alkyl chain length in the anion, the ABSs with cholinium perfluoroalkanoates present well-spaced solubility curves, allowing the conclusion that these ABSs can be tuned by a proper choice of the IL. The phase splitting mechanism was also disclosed through water activity measurements

Levels of mRNA of E1A and E1B over increasing cell culture passages for MDCK-E1 clone #106 (a) and #139 (b).

<p>The gene expression was determined by Real Time reverse transcriptase PCR normalized to the housekeeping gene (GAPDH). Error bars represent a 10% variability error associated with the method.</p

Cell specific viral titers obtained for MDCK-E1 cell clones and the control cell DKZeo at different cell culture passages, in order to assess its effect on the production of CAVGFP.

<p>Low passage corresponds to passage number 20, intermediate to passages between 30–40 and high passage to passages 45–55. Error bars represent a 25% inter-assay variability.</p

Virus amplification in MDCK-E1 clones and DKZeo cells.

<p>The error corresponds to 25% inter-assays variability.</p

Amplification of CAVGFP and JBΔ5 in Cre-expressing clones and the corresponding parental cells.

<p>The error in amplification ratio corresponds to 25% inter-assays variability. The productivity ratio corresponds to the ratio between CAVGFP amplification using Cre expressing and corresponding parental cells. Fold decrease in JBΔ5 production was calculated using the ratio between amplification value of parental cells (MDCK-E1#106 and DKZeo) and the corresponding Cre-expressing cells. Excision efficiency was calculated assuming that the difference in the amplification ratio of parental and corresponding Cre-expressing cells corresponds to unpackaged viral genomes.</p

Half maximal inhibitory concentrations (IC50) of MDCK-E1 and MDCK-E1-Cre cell clones in response to increasing concentrations of <i>tert</i>-butyl hydroperoxide (<i>t</i>-BHP) (causing oxidative stress injury) in the culture medium.

<p>MDCK-E1#121 and MDCK-E1#106 represent high and low E1B expression, while MDCK-E1#106-Cre#10 and MDCK-E1#106-Cre#19 represent high and low Cre-activity, respectively. Error bars correspond to standard-deviation of quadruplicate assays. *<i>p</i><4×10⁻⁵, **<i>p</i><0.05, ***p<0.04, indicating the significance of a single factor Anova analysis.</p

Cell specific productivity and corresponding E1 gene expression of MDCK-E1 cell clones.

<p><b>a.</b> Cell specific infectious viral titer produced in MDCK-E1 clones using DMEM 10% (v/v) FBS and 1% (v/v) NEAA. Error bars represent a 25% inter-assay variability error. <b>b and c.</b> Levels of mRNA E1A (<b>b</b>) and E1B expression (<b>c</b>) obtained for the different MDCK-E1 clones. The gene expression was determined by Real Time reverse transcriptase PCR and normalized to the housekeeping gene (GAPDH). Error bars represent a 10% variability error associated with the method. <b>d.</b> Cell concentration obtained for the different MDCK-E1 clones at virus harvest time. Cells were infected at the same cell concentration. Error bars represent the standard deviation of three independent experiments (n = 3).</p

Cell specific Cre activity of MDCK-E1#106-Cre subclones and DKCre cells measured indirectly through a luciferase assay (see materials and methods).

<p>Error assumes 20% inter-assay variability.</p