After RNA extraction and cDNA synthesis in selected days, miRNA up-regulation was confirmed by miRNA real time PCR and then [gamma]and [beta]chain and GATA-1 expression were investigated by RT and QRT-PCR.
Three and 7 days after transfection, through the use of relative Q-PCR, the [gamma] chain expression increased 3.7, 6.8 and 3.8 folds and GATA-1 expression increased 2.1, 6.0 and 8.0 in comparison with untransfected cells.
Gamma, beta and GATA-1 expression through real-time PCR-based quantification
On the third day after transfection, the gamma ([gamma]), beta ([beta]) and GATA-1 expression were increased 3.7, 0.3 and 2.1 fold, respectively (Figure 2).
On the seventh day after transfection, the gamma, beta and GATA-1 expression were increased 6.8, 0.33 and 6.0 fold, respectively (Figure 2).
Inbred mice, male and female, aged 8-10 weeks, provided in SPF condition by CECAL-FIOCRUZ (Rio de Janeiro), were of the following strains: BALB/c; ALOX (5-LO-deficient) and PAS-129 (wild-type control of the same background) ; and BALB/c mutants lacking an enhancer element in the promoter region of gene coding for the GATA-1 transcription factor , required for eosinophil lineage determination (GATA-1 mice, for short).
Where indicated, naive GATA-1, ALOX, or PAS recipient mice were injected with 1 x [10.sup.6] eosinophils from the appropriate donors (see below) i.p., followed by eotaxin 50 ng/mL, and leukocyte accumulation was monitored in the peritoneal lavage fluid 4 h after eotaxin injection, as above.
Since naive mice carrying a mutation in the high-affinity GATA-1 binding site of the promoter from the gene coding for the GATA-1 transcription factor lack eosinophils , we evaluated the effect of eotaxin on leukocyte numbers 4h after i.p.
We further explored this issue by reconstituting a peritoneal eosinophil population in GATA-1 mice by transfer of purified (90%) BALB/c eosinophils, devoid of neutrophil contamination.
GATA-1 mutations, a transcription factor mutation integral to the normal development of erythroid, megakaryocytic and basophilic cell lines, have been associated with TAM and AML in Down's syndrome, as elucidated by studies done by Tunstall- Pedoe and colleagues.
Abnormalities in the myeloid progenitor compartment in Down's syndrome fetal liver precede acquisition of GATA-1 mutations.
Natural history of GATA-1 mutations in Down syndrome.