title: Characterization of the human artemis promoter by heterologous gene expression in vitro and in vivo.
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.
authors.affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Megan M
authors.initials: MM
authors.lastname: Multhaup
authors.forename: Sweta
authors.initials: S
authors.lastname: Gurram
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Podetz-Pedersen
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Karlen
authors.forename: Debra L
authors.initials: DL
authors.lastname: Swanson
authors.forename: Nikunj V
authors.initials: NV
authors.lastname: Somia
authors.forename: Perry B
authors.initials: PB
authors.lastname: Hackett
authors.forename: Morton J
authors.initials: MJ
authors.lastname: Cowan
authors.forename: R Scott
authors.initials: RS
authors.lastname: McIvor
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001853:Bone Marrow / Q000378:metabolism*; D016026:Bone Marrow Transplantation; D002460:Cell Line; D004260:DNA Repair / Q000235:genetics; D004268:DNA-Binding Proteins; D004720:Endonucleases; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D016086:Lentivirus; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D058990:Molecular Targeted Therapy / Q000379:methods*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016511:Severe Combined Immunodeficiency / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000628:therapy*; D014157:Transcription Factors / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D020648:Peptide Elongation Factor 1; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; C426344:DCLRE1C protein, human; D004720:Endonucleases