II - MATERIALS AND METHODS
A) MATERIALS :
a)
Cellular lines :
Three cellular lines were used :
- Line HL-60 was obtained starting from cells of a patient
reached of a myeloblastic acute leukemia of type 2 according to classification
FAB (French-American-British). Cells HL-60 have the characteristic to be
Bi-potential, i.e. to be different either towards the way granulocytaire, or
towards the way monocytaire, according to whether the ATRA or the VD3 is used
(Breitman and coll, 1980).
- The K562 line was obtained starting from cells of a patient
reached of a chronic leukemia myéloïde. The cellular population was
identified like undifferentiated and of granulocytic series.
- The Cos-6 line comes from a kidney of Monkey.
b) Antibody :
Several antibodies were used : the monoclonal antibody of
anti-myc Mouse (anti myc-TLS, IgG1, Kappa, Rock Molecular Biochemicals), the
antibody polyclonal of Anti-RXR Rabbit (RXR 444, gift of Cecile
Rochette-Egly and Pierre Chambon), the antibody polyclonal of Anti-RAR
Rabbit (RAR 115, gift of Cecile Rochette-Egly and Pierre Chambon) and the
antibody polyclonal of Anti-TLS Rabbit (gift of Francoise Moreau-Gachelin).
c)
Inductive agents :
The ATRA (Hoffmann the Rock) east dissolves in the DMSO with a
concentration of 10-2 M, preserved at- 80°C safe from the light, diluted
in RPMI to obtain a concentration of 10-4 M, stored with- 20°C and used in
the culture media to the final concentration of 10-6 Mr. AR 9-cis carried
out and is in the same way used with the final concentration of 10-6 Mr.
d)
Plasmides :
- The plasmide Tkgal is a plasmide in which is clone ADNc of -
galactosidase under the control of a délété fragment of
promoter TK of the virus of the Herpes (gift of H. of Tea).
- The RARE plasmide is a plasmide pXp2, in which ADNc of the
luciférase is under the control of the promoter of gene RAR2 (inserted
promoter upstream, gift of H. of Tea).
- The vector of expression pcs3-MT (Myc-Tag) contains the
promoter/enhancer cytomegalovirus simien IE94, promoter SP6 and six copies of
the épitope Myc 9e10 (gift of Francoise Moreau-Gachelin).
- pcs3-MT-TLS codes for a protein containing the
épitope Myc and the amino acids of TLS of 1-526 (gift of Francoise
Moreau-Gachelin).
- pcs3-MT-E1A was generated by insertion of the EcoRI-XbaI
fragment of E1A of the plasmide Sp4 in the EcoRI-XbaI site of the vector
pcs3-MT (gift of Francoise Moreau-Gachelin).
- The pSG5-hRAR and the pSG5-mRXR are plasmides of
expression containing ADNc of nuclear receivers RAR and RXR (gift of Cecile
Rochette-Egly and Pierre Chambon).
- The MVC-Luc is a plasmide puC18 of 6 KB, without
«polylinker», in which ADNc of the gene luciférase under the
control of the promoter of the cytomegalovirus is clone, which is a strong
promoter (gift of H. of Tea).
e)
Oligonucléotides :
In the technique of delayed-action on freezing :
DR5: 5 ' - GATCAGGGTTCACCGAAAGTTCACTCGCATATATTAG-3'
(Eurogentec).
In the technique of RT : start RT: 5 ' -
GGAGAGCTTGGGCGACC-3 '(Genset).
In the technique of PCR: start PCR-E1A: 5 ' -
ATTATCTGCCACGGAGGTGT-3': (Genset).
| 
