Spontaneous switch from Agamma- to beta-globin promoter activity in a stable transfected dual reporter vector.
Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for (A)gamma- and beta-globin gene, each one linked to a different Luciferase, in stably transfected GM979 cells for as long as 1-2 years from transfection. The transfected GM979 cells rapidly (within 1 month) evolved into a stable population which expresses constant levels of reporters for more than a year of continuous bulk culture. No silencing of the inserted construct was observed over time. In contrast, after 1 month, the reporter activity (both from (A)gamma- and beta-promoter) expressed per cell increased over time. The analysis of the Luciferase contained in single cell clones indicated that the higher reporter activity was due to increased gene expression per cell rather than to clonal selection of the most expressing clones. Since the activity driven by the beta-promoter increased 10-fold more than that driven by the (A)gamma one, the ratio between (A)gamma-driven/((A)gamma-driven + beta-driven) reporter activity in the cells decreased after 1 month and became similar to the gamma/(gamma + beta) globin mRNA ratio expressed by adult erythroid cells. Moreover, although both cells from early and late bulk culture responded to incubation with butyric acid, a known inducer of fetal globin gene expression, by increasing the reporter activity driven by the (A)gamma-promoter, only cells from late bulk culture decreased, as normal primary erythroblasts do, the activity of the reporter driven by the beta-promoter. These results suggest that the rapid changes in activity driven by the (A)gamma- and beta-globin promoters occurring during the first month after transfection may represent a novel in vitro model to study epigenetic regulation of the (A)gamma- and beta-promoter during the fetal to adult hemoglobin switch and confirm GM979 cells stably transfected with the dual reporter construct as a reliable assay for automated screening of chemical inducers of fetal globin gene activation.
Migliaccio
Giovanni
G
Gene and Cell Therapy, Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italy. migliagi@iss.it
Di Baldassarre
Angela
A
Di Rico
Cristina
C
Di Noia
Antonella
A
Nakamoto
Betty
B
Cao
Hua
H
Skarpidi
Eva
E
Migliaccio
Anna Rita
AR
D000818:Animals; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005319:Fetal Hemoglobin / Q000235:genetics; D017930:Genes, Reporter; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D008156:Luciferases / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic; D014162:Transfection
D005914:Globins; D005319:Fetal Hemoglobin; D008156:Luciferases