CAPITULO III DERECHO ALIMENTARIO
3.1 ANTECEDENTES DE LOS ALIMENTOS.
3.9 PROPORCIONALIDAD DE LA PENSION ALIMENTICIA
To generate recom binant adenoviruses (Ad), the cD NA s for Rho GTPases were subcloned into the admid transfer vectors pCR259 and pCR244 from pcDNA3- N19RhoA, pUHG 10-3N 17Cdc42, pcEX V 3-N 17Racl and pcEX V 3-V 12Rac 1. These vectors express amino-terminal myc-tagged dominant negative RhoA, dominant negative Cdc42, dominant negative R a d and constitutively activated R acl. The N19RhoA mutant was kindly provided by Alan Hall. A fter subcloning genes into the transfer vector (N19RhoA, N17Cdc42 into pCR244, N 17R acl and V 12R acl into pCR259; pCR259 and pCR244 differ only in the m ulticloning site), positive clones were identified by restriction enzyme digestion and subsequently sequenced. Sequence analysis confirmed that in N19RhoA, amino acid 19 was changed from threonine (ACA) to asparagine (AAC), in N17Cdc42 amino acid 17 threonine (ACA) was changed to asparagine (AAT), in N17Racl amino acid 17 threonine (ACT) was changed to asparagine (AAT) and that in V 12R acl amino acid 12 glycine (GGA) was changed to valine (GTA). The admid transfer vectors were transformed into an E.coli K12 strain already transformed with two vectors coding for a transposase and the Ad5 genome (adenovirus homing vector). Thus, transposition of the gene occurred from the transfer vector into the adenovirus homing vector which contained the cytomegalovirus (CMV) promotor concomitantly disrupting
2.2.5.2 Selection of recombinant admids
After transformation, bacteria were plated on Bluo-Gal (Life Technologies)/IPTG plates and only white colonies were selected which in addition had been selected to be chloramphenicol resistant (CmR), tetracycline sensitive (TcS) and ampicillin sensitive (ApS). The plates contained 20 |ig/m l chloramphenicol, 15 )Lig/ml tetracyclin and 100 jig/ml ampicillin. Clones were picked and DNA extracted, retransformed into competent DHIOB E .co li (Gibco BRL, Life Technologies, Paisley, UK) and reselected on chloramphenicol (20 ]Llg/ml) and Bluo-Gal/IPTG plates (20 |Lig/ml). Bluo-Gal, IPTG, tetraycyclin, ampicillin and chloramphenicol were purchased from Sigma, Poole, UK.
2.2.5.3 Transfection o f cells and production of adenoviral master stocks
The recombinant admid DNA was linearized with the restricition enzyme P a d to remove the bacterial promoter and CmR gene. Using lipofectamine reagent (1 mg/ml) (Gibco BRL, Life Technologies, Paisley, UK) 293 cells were transfected with 1 pg admid DNA. The day before transfection, cells were seeded in six well trays (35 mm diameter per well) at a density of 2.5 x 10^ cells/ml per well in 10% FCS/ DMEM. The following day, 1 pg DNA in 100 pi DMEM was combined with 10 pi o f Lipofectamine in 100 pi DMEM and incubated for 1 h at room temperature. Meanwhile, cells were washed twice with serum- free medium to remove FCS. Then 800 pi of DMEM was added to the transfection mix which was then added to the washed cells. The following day, after overnight incubation, the solution was removed and cells were washed twice with serum-free medium before feeding with 2 ml DMEM containing 2% FCS per well.
After further incubation o f transfected 293 cells for 5 days, it was observed that some cells detached from the tissue culture plastic due to production of live virus. The virus was therefore harvested by scraping the attached cells into the medium and then released from cells by three cycles of flash-freeze-thawing. Cellular debris was removed by centrifugation at 2000 g for 10 min. The supernatant was used to infect a nearly confluent 175 cm2 flask (70-80% confluency) of 293 cells. To reach maximal efficiency of infection, the cell medium contained only 2% FCS and the total volume was 10 ml for a 175 cm2 flask. After 1 week o f incubation, cells were detached easily from the tissue culture plastic by shaking. Cells were transferred to a 50 ml tube, centrifuged at 2000 g for 1 0 min, and the pellet containing most virus particles was resuspended in 1 ml of 2%
FCS/DMEM. Virus was isolated by three cycles o f flash-freeze-thawing. The lysate was again centrifugated and the supernatant was used to infect three nearly confluent 175 cm2 flasks of 293 cells.
2.2.5.4 Titration of adenoviruses
When these flasks were harvested after 3 to 4 days, the titer of the adenoviruses was determined by titration on 911 cells. 911 cells were originally generated by transfection of primary human embryonic retina (HER) cells with the DNA coding for the E l gene of the Ad5 serotype. They have been shown to perform well in the plaque assays (CPE assay, cytopathic toxicity assay) as plaques used to determine the viral titer already become apparent in monolayers o f 911 cells 3-4 days after infection versus 4-10 days in monolayers o f 293 cells (Fallaux et al., 1996). The RCA (replication com petent adenovirus) assay was performed on A549 (human lung cancer) cells as they can only propagate replication-competent adenovirus (Zhang, 1997). Cells were set up 24 h before infection in 96 well trays at 10^ cells/well. Serial dilutions o f virus (10'^- 10"^^) were added in 50 jil aliquots to the cells and incubated at 37°C for several days. Plaques were counted using standard bright field microscopy and the viral titer calculated. Viral titers
8 10
(plaque-forming units (pfu)) obtained in 911 cells were in the range of 10 to 10 pfu/ml for the master stocks when plaques were counted by bright-field microscopy.
2.2.5.5 Production of seed stocks
To produce the viral seed stocks, twenty 175 cm^ flasks o f nearly confluent 293 cells were infected at 0 . 0 1 multiplicity of infection (m.o.i) (number of virus particles per cell)
and harvested after 1 week. The low m.o.i was used for the production of the seedstock to reduce the level o f defective interfering particles (DI). Dis are always made in normal adenoviral replication as a by-product. A DI will not lead to production o f complete adenovirus particles, due to the defective genome (Dimmock and Primrose, 1987). The harvested adenovirus was stored in 10% glycerol/DM EM to allow repeated freeze- thawing of the stored aliquots. Stocks were frozen in 1 ml or 100 pi aliquots and were stored for short-term use at -2 0 °C and for long-term use at -7 0 °C. At 4 °C adenovirus is stable for 24 h and no decrease in titer was ever observed by repeated freeze-thawing of the aliquots. In addition, the seed stocks should be stored and subsequent batch stocks should be derived from the seed stock to prevent an increase in RCA and DI.
2.2.5.6 Expression of Rho GTPases in MDCK cells using adenoviruses
N19RhoA, N17Cdc42, N 17R acl and V 12R acl were expressed in M DCK cells by adenovirus-mediated gene transfer. To evaluate the efficiency of adenoviral infection of M DCK cells and the m.o.i required for efficient gene expression after 24 h, a control virus AdCMV-p-Gal was used to visualize infected cells. After three days in culture in 10% FCS/DMEM, the medium was replaced in confluent and subconfluent MDCK cells
with 0.2% FCS/DMEM (infection medium). As it is commonly assumed that high serum inhibits adenoviral infection, medium containing low serum was used for all infection experiments (except for filter-grown monolayers).
Recombinant adenovirus were applied at m.o.i 100 either to subconfluent MDCK cells grown on tissue culture plastic or glass coverslips in 0.2% FCS/DMEM or to the apical surface of M DCK cells grown on Transwell filters in 10% FCS/DMEM for 6 h. After
infection, the medium was replaced with either 0.2% FCS/DMEM for subconfluent or 10% FCS/DMEM for filter-grown MDCK cells with or without 10 ng/ml HGF/SF. Cells were then incubated at 37°C for different timepoints (18 h for immunofluorescence, immunoprécipitation and TER studies, 42 h for TEM).