Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Epigenetic modulation of intestinal Na + /H + exchanger-3 expression

doi: 10.1152/ajpgi.00293.2017

Figure Lengend Snippet: Effect of DNA methylation on Na+/H+ exchanger-3 (NHE3) promoter activity in Caco-2 cells. A: human (h) NHE3 promoter region flanking the area between −1509 and +127 (+1 is the transcription initiation site) was amplified by PCR and cloned in a promoterless cytosine guanine dinucleotide (CpG)-free vector upstream of the lucia gene (pCpG-EV). B: CpG-free EF1 promoter lucia vector (pCpG- hEF1) was used as control for methylation. The NHE3 promoter construct (pCpG-NHE3) and the control vector (pCpG- hEF1) were subjected to in vitro DNA methylation. The methylated and unmethylated control vector and NHE3 promoter construct were transiently transfected in Caco-2 cells along with the mammalian expression vector of β-galactosidase. Posttransfection (48 h), cells were harvested, and lucia activity and β-galactosidase activity were measured. The relative promoter activity was determined as a ratio of lucia to β-galactosidase. Results represent means ± SE of 3–4 separate experiments and are expressed as % of control comparing methylated control vector (pCpG-hEF1 methylated) or methylated NHE3 promoter construct (pCpG-NHE3 methylated) with unmethylated control vector (pCpG-hEF1) or unmethylated NHE3 promoter construct (pCpG-NHE3). **P < 0.01 compared with unmethylated vector/construct as assessed by Student's t-test. C: effect of 5-azacytidine on DNA methylation in the promoter region of NHE3 in Caco-2 cells: DNA was extracted from Caco-2 cells treated with or without 5-azacytidine (10 μM) for 48 h. DNA methylation was determined by qPCR after MethyMiner precipitation using specific primers described in materials and methods, normalized to DNA input, and expressed as fold change. Results represent means ± SE of 3–4 separate experiments. **P < 0.01 compared with untreated cells (control) as assessed by Student's t-test.

Article Snippet: A pCpG-free lucia vector with EF1 promoter (Invivogen) was used as a control for the methylation experiment.

Techniques: DNA Methylation Assay, Activity Assay, Amplification, Clone Assay, Plasmid Preparation, Methylation, Construct, In Vitro, Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation *

doi: 10.1074/jbc.M113.546283

Figure Lengend Snippet: Effect of DNA methylation on NPC1L1 promoter activity. Human NPC1L1 promoter region flanking the area between −1741 and +56 (+1 is the transcription initiation site) was amplified by PCR and cloned into a promoterless CpG free vector (pCpG free basic lucia) upstream of the lucia gene. The NPC1L1 promoter construct as well as the empty vector were then subjected to in vitro methylation. The methylated and unmethylated empty vector (pEV) and NPC1L1 promoter construct (L1p) were transiently transfected into Caco2 cells along with the mammalian expression vector of β-galactosidase. 48 h post-transfection, cells were harvested, and lucia activity and β-galactosidase activity were measured. The relative promoter activity was determined as a ratio of lucia to β-galactosidase (A). Data are presented as fold increase compared with unmethylated empty vector and represent mean ± S.E. of three independent experiments performed on separate occasions. *, p < 0.05 compared with empty vector pEV. CpG free EF1 promoter lucia vector (pCpG free lucia) was used as control for methylation as described under “Results” (B). Data are presented as fold change relative to unmethylated vector.

Article Snippet: For the construction of pCpG free L1 vector, the DNA fragment containing −1741/+56 region of the human NPC1L1 promoter was amplified using forward 5-ATCGATGCGAGTACTTGGACTCTATCTCTCTGTGG-3′ and reverse 5-ATCGATGCGAAGCTTCCCAGGTCTGGGAAGGGGTCA-3′ primers, and the amplified promoter fragments were then inserted into a promoterless CpG free vector (pCpG free basic lucia, InvivoGen, San Diego) upstream of the lucia reporter gene.

Techniques: DNA Methylation Assay, Activity Assay, Amplification, Clone Assay, Plasmid Preparation, Construct, In Vitro, Methylation, Transfection, Expressing, 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: 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: To assess the vector delivery using pCpG-free lacZ reporter gene, CD-1 female mice of 6–8 weeks of age (Charles River, UK), weighing 28–30 g, were confined in the same Plexiglas box mentioned above.

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: To assess the vector delivery using pCpG-free lacZ reporter gene, CD-1 female mice of 6–8 weeks of age (Charles River, UK), weighing 28–30 g, were confined in the same Plexiglas box mentioned above.

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