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Updated: Aug 9, 2026
Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation
Published on: June 21, 2016
Endogenous CRX expression and IRBP promoter activity in retinoblastoma cells
J H Boatright1, D E Borst, E Stodulkova
1Emory Eye Center, Atlanta, GA, USA. jboatri@emory.edu
Purpose:
To determine whether antisense oligonucleotides (AODNs) targeted against CRX, a photoreceptor-specific trans-acting factor, suppress CRX expression and interphotoreceptor retinoid binding protein (IRBP) promoter activity.
Methods:
Cultures of human retinoblastoma cells were transfected with chloramphenicol acetyltransferase (CAT) reporter plasmids containing a mouse IRBP promoter and AODNs directed against CRX. RT-PCR using primers specific to CRX, OTX2, GAPDH, or RNase H was conducted on total RNA isolated from retinoblastoma cells at various times following transfection with AODNs.
Results:
Transfection of retinoblastoma cells with IRBP promoter CAT constructs alone produced high activity. Co-transfection with AODNs suppressed IRBP promoter activity in a concentration-dependent manner, with half-maximal effect produced at about 2 nM AODN concentration. Transfection with CAT constructs containing an SV40 promoter produced high activity that was unaffected by co-transfection with AODNs. RT-PCR products were obtained for all target sequences. CRX RT-PCR product from cells transfected with AODNs was greatly diminished following transfection with an AODN whereas control transcripts, including that of OTX2, were relatively unaffected.
Conclusions:
The CRX-specific AODNs specifically and potently suppressed CRX expression and IRBP promoter activity, as measured by RT-PCR and transient transfection assays, respectively. Little or no effect was seen on controls. These data suggest that endogenous CRX is required for IRBP promoter activity in retinoblastoma cells.
