MATERIALS AND METHODS
Mice, parasites and cells.
All animal care and procedures were performed in accordance with institutional guidelines and European regulations. Experiments were conducted using AQP9-/- and their litternate control aged from 7 to 10 weeks. P. yoelii (265BY) P. berghei (ANKA strain) and P. falciparum (NF54 strain) sporozoites were obtained by dissection of infectedAnopheles stephensi mosquito salivary glands.
Human hepatocarcinoma cell lines HepG2-A16-hCD81EGFP (Yalaoui et al 2008 plos pathogens), HepG2-CD81 (Bartosh et al 2003 JBC) and Huh-7 were cultured in DMEM (Invitrogen) supplemented with 10% FCS, 2 mM glutamine and antibiotics (Invitrogen, Gibco).
Primary mouse hepatocytes were isolated as previously described (Renia et al., 1990), seeded at a density of 4x 104 cells per well in 96-well microplates and cultured at 37 °C in 5% CO2 in William’s E medium (Invitrogen, Gibco) supplemented with 2% penicillin-streptomycin, 1% sodium pyruvate, 1% L-glutamine, 1% insulin-transferrin-selenium, 1% non essentials amino acids and 10% fetal bovine serum (Invitrogen, Gibco).
Human liver fragments were collected following informed consent provided from patientsundergoing a partial hepatectomy. The collection and use of these tissues were undertaken in accordance with French government ethical regulations. Primary human hepatocytes were isolated from healthy parts of the liver biopsies as previously described (Carraz et al., 2006), seeded at a density of 8 x 104 cells in 96-well microplates precoated with rat tail collagen I (Becton Dickinson, Le Pont deClaix, France) and cultured at 37 °C in 5% CO2, in the same culture medium as above, supplemented with 10–7 M dexamethasone (Sigma, Saint Quentin Fallavier, France). Mouse and human hepatocytes were cultured for 24 h before silencing with siRNA.
Small interfering RNA and lentiviral transduction.
We used small double stranded RNA oligonucleotides targeting human CD81 (5’- GCA CCA AGT GCA TCAAGT A -3’), human AQP3 (Qiagen HP validated siRNA AQP3_5), human AQP9 (5’- CTG CTG ATC GTG GGA GAA A-3’ and 4 Qiagen HP GenomeWide siRNA). A siRNA oligonucleotide targeting human CD92 (5’- AAG GCA AGA ACT GAA AAC T -3’) was used as a control siRNA throughout the study. Primary hepatocytes were transfected with 30nM of siRNA with Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’srecommendations. Following siRNA transfection, cells were cultured for 72 hours before western blot, flow cytometry analysis or sporozoite infection.
The TRIPΔ3-CMV-AQP9 plasmid was constructed by inserting the human AQP9 cDNA sequence in the TRIPΔ3-CMV vector (Ref). Vector particles were produced by transient calcium phosphate cotransfection of 293T cells by the TRIPΔ3-CMV-AQP9 plasmid, anencapsidation plasmid and a vesicular stomatitis virus envelope expression plasmid, as described. Cells were transduced by incubating 1/1000 of concentrated viral preparation 3h before washing and viral particles expressing GFP were used as a control.
(Note personnelle Patrick; J-3+Si => J-2lavage+DMSO (cad élimination des Si => J0 spz => J3->5 schizontes => => il n’y a pas de prolif avec HH => pas oupeu de synthèse de protéines; cependant, il est possible qu’il y ait une néosynthèse de prot => problème pour interpréter le rôle des protéines sur développement/maturation.
Real-time PCR quantification of liver-stage parasite burden.
Wild-type (n = 4) and AQP91-/- (n = 4) male mice were infected by retro-orbital injection with 4 x 104 P. berghei ANKA-GFP sporozoites and sacrificed 40h postinoculation. Livers were harvested, homogenized using a Polytron homogenizer and total RNA was isolated using the Micro-to-midi Total RNA purification system (Invitrogen). The detection and quantification of the liver stage parasite load was performed on the corresponding total cDNAs, as detailed previously (Rodrigues et al., 2008). Amplified P. berghei 18S sequences were normalized to mouse...
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