Dioctadecyldimethylammonium:monoolein nanocarriers for efficient in vitro gene silencing

This study describes a novel liposomal formulation for siRNA delivery, based on the mixture of the neutral lipid monoolein (MO) and cationic lipids of the dioctadecyldimethylammonium (DODA) family. The cationic lipids dioctadecyldimethylammonium bromide (DODAB) and chloride (DODAC) were compared in order to identify which one will most efficiently induce gene silencing. MO has a fluidizing effect on DODAC and DODAB liposomes, although it was more homogeneously distributed in DODAC bilayers. All MO-based liposomal formulations were able to efficiently encapsulate siRNA. Stable lipoplexes of small size (100-160 nm) with a positive surface charge (>+45 mV) were formed. A more uniform MO incorporation in DODAC:MO may explain an increase of the fusogenic potential of these liposomes. The siRNA-lipoplexes were readily internalized by human nonsmall cell lung carcinoma (H1299) cells, in an energy dependent process. DODAB:MO nanocarriers showed a higher internalization efficiency in comparison to DODAC:MO lipoplexes, and were also more efficient in promoting gene silencing. MO had a similar gene silencing ability as the commonly used helper lipid 1,2-dioleyl-3-phosphatidylethanolamine (DOPE), but with much lower cytotoxicity. Taking in consideration all the results presented, DODAB:MO liposomes are the most promising tested formulation for systemic siRNA delivery.This work was supported by FEDER through POFC - COMPETE and by national funds from FCT through the projects PEst-C/BIA/UI4050/2011 (CBM.A), PEst-C/FIS/UI0607/2011 (CFUM), and PTDC/QUI/69795/2006, while Ana Oliveira holds scholarship SFRH/BD/68588/2010. Eloi Feitosa thanks FAPESP (2011/03566-0) and CNPq (303030/2012-7), and Renata D. Adati thanks FAPESP for scholarship (2011/07414-0). K. Raemdonck is a postdoctoral fellow of the Research Foundation - Flanders (FWO-Vlaanderen). We acknowledge NanoDelivery-I&D em Bionanotecnologia, Lda. for access to their equipment

Direct interaction between hnRNP-M and CDC5L/PLRG1 proteins affects alternative splice site choice

International audienceHeterogeneous nuclear ribonucleoprotein-M (hnRNP-M) is an abundant nuclear protein that binds to pre-mRNA and is a component of the spliceosome complex. A direct interaction was detected in vivo between hnRNP-M and the human spliceosome proteins cell division cycle 5-like (CDC5L) and pleiotropic regulator 1 (PLRG1) that was inhibited during the heat-shock stress response. A central region in hnRNP-M is required for interaction with CDC5L/PLRG1. hnRNP-M affects both 5 0 and 3 0 alternative splice site choices, and an hnRNP-M mutant lacking the CDC5L/PLRG1 interaction domain is unable to modulate alternative splicing of an adeno-E1A mini-gene substrate

(A) Cell extracts prepared from 293T cells were incubated with either a mouse monoclonal anti–U1 70K antibody bound to Sepharose beads (lanes 2 and 3) or Sepharose beads alone (lanes 4 and 5)

The bound proteins were analyzed by Western blotting with anti-SF2/ASF antibody. Alternatively, the assay was performed in the presence of RNase (lanes 3 and 5). (B) In vivo detection of protein–protein interactions between ECFP-U1 70K and EYFP-SF2/ASF by FRET acceptor photobleaching microscopy. HeLa cells coexpressing ECFP-U1 70K and EYFP-SF2/ASF were analyzed on a wide-field fluorescent microscope. Images were acquired before and after photobleaching. A nonbleached region similar to the bleached region (arrows) was included in the data analysis for comparison. Bars, 15 μm. (C) Donor and acceptor mean fluorescence intensities monitored in the bleached and nonbleached regions were plotted over time. (D) FRET efficiencies for the interaction between ECFP-U1 70K and EYFP-SF2/ASF in the presence and absence of DRB. A FRET efficiency for these interactions was calculated as described in Materials and methods and, when >5%, was considered significant. Plot is of FRET efficiencies ± SD (mean for 8–27 cells) between ECFP + EYFP pairs before and after DRB treatment. P-values were obtained from the two-tailed homoscedastic test comparing the FRET efficiencies with and without DRB treatment.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p

(A) HeLa cells were cotransfected with EGFP-U2AF35 and either mCherry-C1 or mCherry-SF2/ASF

Confocal images are of transfected cells and FLIM images of the same cells, in which FRET efficiency and FRET amplitude are shown in pseudocolor. The color scale with the respective efficiency (%) is indicated. Top, EGFP-U2AF35 + mCherry-C1; Middle, EGFP-U2AF35 + mCherry-SF2/ASF; Bottom, EGFP-U2AF35 + mCherry-SF2/ASF in the presence of DRB. Bars, 10 μm. (B) FRET efficiencies determined by FLIM for interaction of SF2/ASF with U2AF35 in the presence and absence of DRB. Plot is of mean FRET efficiencies ± SD for seven to nine cells. To measure the FRET efficiency in the speckles and nucleoplasm, a region characteristic of each was selected for each cell. P-values were obtained as described in the legend. *, P < 0.1.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p

(A) HeLa cells were transfected with EGFP–U1 70K and cotransfected with either mCherry-C1 or mCherry-SF2/ASF

Shown are confocal images of transfected cells and FLIM images of the same cells, in which mean fluorescence lifetime is shown in pseudocolor. The color scale with the respective lifetimes (in picoseconds [ps]) is indicated. The percentage of FRET efficiencies and FRET amplitude are shown in continuous pseudocolor. The color scale with the respective FRET efficiencies (percentage) is indicated. The FRET amplitude % represents the fraction of interacting donor molecules, also defined as the FRET population % (or concentration of FRET species). (B) FRET between U1 70K and SF2/ASF, in the presence of DRB, measured by FLIM. Experiments were performed exactly as described for A, except cells were treated with 25 μg/ml of DRB for 2 h before images were taken. Bars, 10 μm. (C) FRET efficiencies calculated from FLIM measurements for the interaction of SF2/ASF with U1 70K in the presence and absence of DRB. Plot is of mean FRET efficiencies ± SD for 9–20 cells. To measure the FRET efficiency in the speckles and nucleoplasm, a region characteristic of each was selected for each cell. P-values were obtained as described in the legend. *, P < 0.1.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p

(A) Extracts prepared from 293T cells transiently transfected with either EGFP-U2AF35 and pCG-T7-HCC1

4 (lanes 3 and 4) or EGFP-U2AF35 (lanes 5 and 6) were incubated with anti-T7 antibody bound to Sepharose beads. The bound proteins were analyzed by Western blotting with anti-GFP antibody. Alternatively, the assay was performed in the presence of RNase (lanes 4 and 6). (B) U2AF65 interacts with HCC1 in cultured mammalian cells. Extracts prepared from 293T were incubated with either anti-HCC1 antibody bound to Sepharose beads (lanes 2 and 4) or Sepharose beads alone (lanes 3 and 5). The bound proteins were analyzed by Western blotting with anti-U2AF65 antibody. Alternatively, the immunoprecipitate was treated with RNase (lanes 4 and 5). (C) Effect of DRB on interactions of HCC1 with U2AF35 and U2AF65. Plot is of FRET efficiencies ± SD (mean for 8–18 cells) between ECFP and EYFP fusion proteins measured by FRET acceptor photobleaching. P-values were obtained as described in the legend.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p