)
Epissage in vivo
The cells are collected 24 hours after the transfection.
Co-transfection with the plasmide pCMV-luciférase allows the
standardization of the RT according to the differences observed in the
effectiveness of transfection. A aliquotée fraction of these cells is
used to measure the activity luciférase and the remainder is used to
carry out a total extraction of ARNs. ARNs totals are prepared starting from
the cells by using the protocol of purification of ARN and according to the
instructions of the supplier (RNeasy, Qiagen) and treated twice by Dnase I
(Qiagen). The quality of ARNs obtained is observed by migration on a gel of
agarose denaturing agent. The isoformes of ARN E1A are analyzed by RT-PCR as
described previously (Hallier and coll, 1998). One microgram of ARN retro-is
transcribed by SuperscriptII transcriptase reverse of the virus of the murine
leukemia of Moloney (Gibco-BRL) in the presence of 50 uM of dNTP and 2
picomoles of preceding 3 ' RT E1A in a solution of 8 ul. Approximately a tenth
of ADNc is used for the reaction of amplification (PCR) carried out with
Taq polymerase ADN (Perkin Elmer) in the presence of the starter 5 ' E1A
marked at its end 5 ' by T4 polynucléotide kinase (Boehringer) and of
the ATP P32 (Amersham) like previously described (Hallier and coll, 1998). The
minimum of cycles PCR is used (18 to 22 cycles) in order to detect the signal
in a window of adequate detection. The reactions of control of the RT-PCR
contain a matrix ARN not having undergone an opposite transcription. The
products of the RT-PCR of E1A are put to migrate on a urea/polyacrylamide gel
6% denaturing agent, autoradiographies and quantified with a Biorad system. The
form of proteins coded by the plasmides is checked by the technique of the Blot
Western carried out on samples of transfectées cells from which come
ARNs totals.
e)
Test of transactivation
The transfection day before, the Cos-6 cells are put out of
well, in 5ml of DMEM-6% SVF décomplémenté at a rate of
0,6x106 per well. The next day, the medium is changed for 4 ml of fresh medium.
In each well, is added a beforehand incubated solution of 1 ml with ambient
temperature including/understanding a plasmidic solution (TKGal,
Rare-Luciférase,...), 450 water ug Bi-distilled in addition to 50 ul of
CaCl2 (2,5 M) and 500 ul of HBS 2X (NaCl 280 mm, KCl 10 mm, Na2HPO4 1,2 mm,
Glucose 12 mm, Hépès 50 mm).
Cells HL-60 are électroporées with an equivalent
plasmidic solution.
After a 12 hours incubation to 37oC, the cells are washed 2
times at the PBS 1X. A plug of lysis (200 ul) is added in each well and the
cells are separated using a scraper. The cells are recovered in tubes
Eppendorff, are vortexées and centrifuged 2 min. with 12000g and 4oC.
The activity of the luciférase (according to the protocol of Luciferase
Assay System, Promega, Madison, EUA) in the lysate is measured in a luminometer
and is expressed in arbitrary unit. The activity of - Galactosidase (according
to the protocol of BM Chemiluminescence ELISA Substrate - Gall, Boehringer
Mannheim, Mannheim, the Federal Republic of Germany) is also measured in order
to standardize measurements obtained of the luciférase activity.
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