Plasmid psGFP2-C1 (Addgene Plasmid #22881) was digested with AseI and AgeI restriction enzymes (New England BioLabs) to remove the cytomegalovirus (CMV) enhancer and promoter sequence. A region of approximately 2 kb upstream of the CD37 transcription start site (chr19:49 836,812-49 838,766 [GRCh37/hg19]) was amplified from genomic DNA of SU-DHL-5 cells (supplemental Table 2) and ligated into digested psGFP2-C1 to replace the CMV enhancer and promoter region. Cell lines were cotransfected with CMV promoter-GFP (green fluorescent protein) or CD37 promoter-GFP construct and pmScarlet-C1 (Addgene plasmid #85042) as the transfection loading control. In case of cotransfection of the CD37 promoter-GFP with PU.1 and/or IRF2 (both in pCDNA3.1, Genscript), pIRF670-N1 plasmid22 (link) was used as a loading control. If required, pCDNA3.1 empty vector was added to obtain equal amounts of total plasmid DNA. Fluorescent protein expression was analyzed by flow cytometry 24 hours after transfection. Viable cells were gated on positive expression of Scarlet and/or GFP, and the percentage of GFP-expressing cells within this population was determined.
Protocol detail
Cloning CD37 Promoter-Driven GFP Construct
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Cell lines
Cytomegalovirus
Flow cytometry
Genomic
Irf2
Plasmid
Protein
Restriction enzymes
Transcription start site
Transfection
Vector
Corresponding Organization :
Other organizations : National Center for Tumor Diseases, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Radboud University Medical Center, Oncode Institute, Canisius-Wilhelmina Ziekenhuis, Rijnstate Hospital, Duke Medical Center, Amsterdam University Medical Centers
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Variable analysis
independent variables
- Cotransfection of CD37 promoter-GFP construct with PU.1 and/or IRF2
- Fluorescent protein (GFP) expression analyzed by flow cytometry
- Cotransfection of CMV promoter-GFP (green fluorescent protein) construct
- Cotransfection of pmScarlet-C1 plasmid as the transfection loading control
- Use of pIRF670-N1 plasmid as a loading control for cotransfection of CD37 promoter-GFP with PU.1 and/or IRF2
- Addition of pCDNA3.1 empty vector to obtain equal amounts of total plasmid DNA
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