plasmid pcpg free lucia pcpg lucia  (InvivoGen)

Journal: PLoS ONE

Article Title: Nebulisation of Receptor-Targeted Nanocomplexes for Gene Delivery to the Airway Epithelium

doi: 10.1371/journal.pone.0026768

Figure Lengend Snippet: A and B Transfections of 16HBE14o- and CFBE41o- cells mediated by RTNs nebulised through the NGI: RTNs were made in clinical grade H 2 O at weight ratio of 1.5∶4∶1 of DHDTMA/DOPE (1∶1 molar ratio), peptide E and pCpG-free lacZ plasmid DNA, respectively. 3 ml of RTN suspension was used for each nebulisation and all three nebulisers were operated at a flow rate of 15 L/min for 10 min. After aerosolisation nebulised nanocomplexes, deposited in the different stages of the NGI, were collected by adding to each stage 1 ml of water. A volume of the collected samples, diluted in OptiMEM, was added onto cells. The expression of the reporter gene was assessed 48 h after delivery. Representative results of transfection experiments in both cell lines for each nebuliser (AeroEclipse II BAN, PARI-LC Plus and Aeroneb® Pro) are shown. The error bars represent the standard error of the mean of quadruplicate wells. Nebulisations through the NGI were repeated 3 times with each nebuliser for both cell lines and cumulative results are reported in . RLU = Relative Light Unit; Thr = throat; S1–S8 = stages 1–8 of the NGI, S8 = micro-orifice collector. C Physicochemical properties pre- and post-nebulisation: The size and the ζ (electrokinetic or zeta) potential were measured using a Malvern Nano ZS Zetasizer on aliquots of pre-nebulised nanocomplexes, of nebulised samples collected in a 50 ml tube, and of residual RTN suspension remaining in the nebuliser chamber upon completion of the nebulisation. The average values of three independent measurements of each sample were automatically performed. Bars represent the average values of the sizes of two independent experiments ± the standard deviation whereas the diamonds indicate the ζ potentials in the same samples ± the standard deviation. PN = pre-nebulisation RTN suspension; post = post-nebulised nanocomplex suspension; LO = (leftover) RTNs retained in the nebuliser chamber. D In vivo delivery of RTNs containing luciferase reporter gene in C57BL6 mice: 2 ml of RTNs, prepared with pCILuc plasmid, were nebulised using either the Aeroneb® Pro or the AeroEclipse II BAN, into a Plexiglas chamber in which 9 mice (C57BL6 strain) were confined. 24 h later, 7 of the murine lungs were then analysed for luciferase expression as described in the methods. Luciferase activity is expressed as Relative light Unit (RLU) per milligram of protein. There was not statistical difference between the two groups when the readings were normalised to protein content, but there was a statistical difference (p<0.05) in the luciferase activity (Mann-Whitney test). E and F Localisation of luciferase expression by immunohistochemistry: Trachea of C57BL6, following a single nebulisation of 2 ml of RTNs containing pCILuc were stained with 3,3′-diaminobenzadine (F), and counterstained with haematoxylin. The transfected tissue was localised in the ciliated airway epithelium whereas the negative control section (E) was not probed with the primary antibody. Objective magnification: 40×.

Article Snippet: The pCpG-free lacZ plasmid (Invivogen, San Diego, CA, USA) encodes for β-galactosidase under the EF1α promoter, but is devoid of CpG dinucleotides.

Techniques: Transfection, Plasmid Preparation, Suspension, Expressing, Zeta Potential Analyzer, Standard Deviation, In Vivo, Luciferase, Activity Assay, MANN-WHITNEY, Immunohistochemistry, Staining, Negative Control

Journal: PLoS ONE

Article Title: Nebulisation of Receptor-Targeted Nanocomplexes for Gene Delivery to the Airway Epithelium

doi: 10.1371/journal.pone.0026768

Figure Lengend Snippet: RTN suspension (6 ml), at a concentration of 160 µg/ml of DNA, was nebulised using the AeroEclipse II BAN in a plexiglass box containing 6 CD1 mice. After 48 h following the nebulisation, the animals were culled and the lungs and the tracheas removed. In the harvested tissues, the levels of β-galactosidase were measured by quantifying the enzymatic activity and by immunodetecting the protein. Control animals were nebulised with the same clinical grade H 2 O in which nanocomplexes were made. A and B Determination of β-galactosidase enzymatic activity: The β-galactosidase activity was measured by CPRG assay in both whole lung lysates (A) and in those from the tracheas (B). The activity of enzyme (mU) in the lysates was calculated against the β-galactosidase activity of Escherichia coli and normalised for the total protein content. (*) in panel B (tracheas) indicates statistically significant difference (Mann-Whitney test) at 0.05 level (p<0.05). No difference was found in panel A, although there the protein activity showed a similar trend. C and D Detection of β-galactosidase protein: The lysates from the whole lungs (C) and the trachea (D) from each mouse were spotted onto PVDF membranes. Dot blots were first probed with goat polyclonal anti-β-galactosidase antibody and then with horseradish peroxidase-labelled rabbit polyclonal anti-goat immunoglobulin. The quantification of duplicate dots per each mouse is shown. The difference between the control group (nebulised with water) and the cohort which received RTNs formed using pCpG-free lacZ reporter gene, was statistically significant at the Mann-Whitney test (*, p<0.05).

Article Snippet: The pCpG-free lacZ plasmid (Invivogen, San Diego, CA, USA) encodes for β-galactosidase under the EF1α promoter, but is devoid of CpG dinucleotides.

Techniques: Suspension, Concentration Assay, Activity Assay, MANN-WHITNEY