Development and characterization of cellular biosensors for HTS of erythroid differentiation inducers targeting the transcriptional activity of γ-globin and β-globin gene promoters

  • Giulia Breveglieri
  • Francesca Salvatori
  • Alessia Finotti
  • Lucia Carmela Cosenza
  • Cristina Zuccato
  • Nicoletta Bianchi
  • Laura Breda
  • Stefano Rivella
  • Alberto Bresciani
  • Monica Bisbocci
  • Monica Borgatti
  • Roberto GambariEmail author
Research Paper
Part of the following topical collections:
  1. New Developments in Biosensors


There is a general agreement that pharmacologically mediated stimulation of human γ-globin gene expression and increase of production of fetal hemoglobin (HbF) is a potential therapeutic approach in the experimental therapy of β-thalassemia and sickle cell anemia. Here, we report the development and characterization of cellular biosensors carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and β-globin gene promoters, respectively; these dual-reporter cell lines are suitable to identify the induction ability of screened compounds on the transcription in erythroid cells of γ-globin and β-globin genes by FACS with efficiency and reproducibility. Our experimental system allows to identify (a) HbF inducers stimulating to different extent the activity of the γ-globin gene promoter and (b) molecules that stimulate also the activity of the β-globin gene promoter. A good correlation does exist between the results obtained by using the EGFP/RFP clones and experiments performed on erythroid precursor cells from β-thalassemic patients, confirming that this experimental system can be employed for high-throughput screening (HTS) analysis. Finally, we have demonstrated that this dual-reporter cell line can be used for HTS in 384-well plate, in order to identify novel HbF inducers for the therapy of β-thalassemia and sickle cell anemia.

Graphical abstract


HbF inducers Thalassemia Reporter systems HTS Erythroid differentiation Sickle cell anemia 



Fetal hemoglobin


Sickle cell anemia


Erythroid precursor cell


Green fluorescence protein


Enhanced green fluorescence protein


Red fluorescence protein


Locus control region


Fluorescence activated cell sorting


Polymerase chain reaction


High-throughput screening





Roberto Gambari is funded by Fondazione Cariparo (Cassa di Risparmio di Padova e Rovigo), CIB (Consorzio Interuniversitario per le Biotecnologie), UE THALAMOSS Project (Thalassemia Modular Stratification System for Personalized Therapy of Β-Thalassemia; n. 306201-FP7-HEALTH-2012-INNOVATION-1), by Wellcome Trust (Innovator Award 208872/Z/17/Z) and by AIFA (AIFA-2016-02364887). This research activity was also supported by Associazione Veneta per la Lotta alla Talassemia (AVLT), Rovigo.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

The β-thalassemia patients have been recruited at Thalassemic Day Hospital (DHT) of the University Hospital “Sant’Anna” (Ferrara, Italy). The approval of the collection and processing of the human biological samples for this research was carried out by the Ethics Committee of Ferrara District, number 06/2013 (approved on June 20, 2013). The study complies with the Declaration of Helsinki, the principles of Good Clinical Practice, and all further applicable regulations in accordance with the ethical standards. All samples of peripheral blood have been obtained after written documentation of informed consent from patient or legal representative. Copies of the consents have been collected for archiving by Thalassemic Day Hospital (DHT) of the University Hospital “Sant’Anna” (Ferrara, Italy).

Supplementary material

216_2019_1959_MOESM1_ESM.pdf (4.4 mb)
ESM 1 (PDF 4.42 MB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Giulia Breveglieri
    • 1
    • 2
  • Francesca Salvatori
    • 3
  • Alessia Finotti
    • 1
  • Lucia Carmela Cosenza
    • 1
    • 2
  • Cristina Zuccato
    • 1
  • Nicoletta Bianchi
    • 4
  • Laura Breda
    • 5
  • Stefano Rivella
    • 5
  • Alberto Bresciani
    • 6
  • Monica Bisbocci
    • 6
  • Monica Borgatti
    • 1
    • 2
  • Roberto Gambari
    • 1
    Email author
  1. 1.Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular BiologyUniversity of FerraraFerraraItaly
  2. 2.Biotechnology CenterUniversity of FerraraFerraraItaly
  3. 3.Department of Chemical and Pharmaceutical SciencesUniversity of FerraraFerraraItaly
  4. 4.Department of Biomedical Sciences and Specialist Surgery, Section of Biochemistry, Molecular Biology and Medical GeneticsUniversity of FerraraFerraraItaly
  5. 5.Hematology DivisionChildren’s Hospital of PhiladelphiaPhiladelphiaUSA
  6. 6.IRBM Science Park SpAPomezia (Rome)Italy

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