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.
Tsuchiya
T
T
Institute of Medical Science, University of Tokyo, Japan.
Ochiai
H
H
Imajoh-Ohmi
S
S
Ueda
M
M
Suda
T
T
Nakamura
M
M
Kanegasaki
S
S
D000740:Anemia / Q000503:physiopathology*; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000502:physiology*; D003001:Cloning, Molecular; D004921:Erythropoietin / Q000235:genetics*; D006801:Humans; D007438:Introns; D007668:Kidney / Q000502:physiology / Q000503:physiopathology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D012016:Reference Values; D013698:Templates, Genetic; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D004921:Erythropoietin