Single-copy transduction and expression of human γ-globin in K562 erythroleukemia cells using recombinant adeno-associated virus vectors: The effect of mutations in NF-E2 and GATA-1 binding motifs within the hypersensitivity site 2 enhancer

Jeffery L. Miller, Christopher E. Walsh, Paul A. Ney, Richard Jude Samulski, Arthur W. Nienhuis

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43 Scopus citations

Abstract

The use of recombinant adeno-associated virus (rAAV) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. We linked hypersensitivity site 2 (HS2) from the locus control region (LCR) to a Aγ-globin gene (Aγ*) mutationally marked to allow its transcript to be distinguished from endogenous γ-globin mRNA. The vector also contains the phosphotransferase gene that confers resistance to neomycin (Neo®). HS2 region mutations within the NF-E2 motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the GATA-1 motif prevented binding of the factor. Several Neo® K562 clones containing a single unrearranged RAAV genome with the Aγ* gene linked to the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed Aγ* mRNA at the same level as the WT clones, compared with a lack of Aγ* signal in the (-)HS2 clones. However, hemin induction of mut-NFE2 clones did not result in an increase in the Aγ* signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the Aγ* mRNA at the same level as WT clones in both uninduced and induced cells. Thus, GATA-1 binding to this site does not appear to be required for the enhancing function of HS2 in this context. This single-copy rAAV transduction model is useful for evaluating the effects of specific mutations in regulatory elements on the transcription of linked genes. This is a US government work. There are no restrictions on its use.

Original languageEnglish
Pages (from-to)1900-1906
Number of pages7
JournalBlood
Volume82
Issue number6
StatePublished - 15 Sep 1993

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