In vitro reconstitution of an erythropoietin gene transcription system using its 5′-flanking sequence and a nuclear extract from anemic kidney

We have developed an in vitro transcription system for the erythropoietin (Epo) gene. This system uses a plasmid carrying 0.2 kb of 5′-flanking sequence from the human Epo gene, rNTPs and a nuclear extract from mouse kidney. The transcribed RNA was assayed by primer extension with an end-labeled primer complementary to the sequence of the plasmid, dNTPs and reverse transcriptase. The primer extension product corresponding to the transcript was detected on a sequencing gel. The in vitro promoter activity of the Epo 5′-flanking sequence was observed with a nuclear extract from anemic kidney but not with that from normal kidney.