Article

Characterization of the human artemis promoter by heterologous gene expression in vitro and in vivo.

Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, USA.
DNA and cell biology (impact factor: 2.28). 06/2011; 30(10):751-61. DOI:10.1089/dna.2011.1244 pp.751-61
Source: PubMed

ABSTRACT Artemis is an endonucleolytic enzyme involved in nonhomologous double-strand break repair and V(D)J recombination. Deficiency of Artemis results in a B- T- radiosensitive severe combined immunodeficiency, which may potentially be treatable by Artemis gene transfer into hematopoietic stem cells. However, we recently found that overexpression of Artemis after lentiviral transduction resulted in global DNA damage and increased apoptosis. These results imply the necessity of effecting natural levels of Artemis expression, so we isolated a 1 kilobase DNA sequence upstream of the human Artemis gene to recover and characterize the Artemis promoter (APro). The sequence includes numerous potential transcription factor-binding sites, and several transcriptional start sites were mapped by 5' rapid amplification of cDNA ends. APro and deletion constructs conferred significant reporter gene expression in vitro that was markedly reduced in comparison to expression regulated by the human elongation factor 1-α promoter. Ex vivo lentiviral transduction of an APro-regulated green fluorescent protein (GFP) construct in mouse marrow supported GFP expression throughout hematopoeitic lineages in primary transplant recipients and was sustained in secondary recipients. The human Artemis promoter thus provides sustained and moderate levels of gene expression that will be of significant utility for therapeutic gene transfer into hematopoeitic stem cells.

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Keywords

1 kilobase DNA sequence upstream
 
5' rapid amplification
 
APro-regulated green fluorescent protein
 
Artemis expression
 
Artemis gene transfer
 
Artemis results
 
effecting natural levels
 
Ex vivo lentiviral transduction
 
gene expression
 
GFP expression
 
global DNA damage
 
human Artemis gene
 
human Artemis promoter
 
moderate levels
 
nonhomologous double-strand break
 
overexpression
 
significant reporter gene expression
 
therapeutic gene transfer
 
transcriptional start sites
 
V(D)J recombination