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Gene expression associated with lentiviral expression of PRAME in normal hematopoietic progenitors

ABSTRACT: To identify gene expression that distinguishes hematopoietic cells that express PRAME from those that do not, normal CD34+ cells with forced PRAME expression were compared to cells without PRAME expression in culture over time (days 4, 7, 14) using Affymetrix HU-133A microarrays Normal CD34+ progenitor cells not expressing PRAME were transduced with either PRAME cDNA (no 3' utr) or a corresponding empty vector control by lentiviral vector infection. Two replicates were performed for each condition (PRAME and control) at three experimental time points (days 4, 7, and 14 in culture). A single-stranded linear amplification protocol was used to process total RNA (one microgram) for the microarrays. This protocols is described in detail in PMID 14706461.

ORGANISM(S): Homo sapiens

SUBMITTER: Oehler VG

PROVIDER: S-ECPF-GEOD-17100 | biostudies-other | 2009 Oct

REPOSITORIES: biostudies-other

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Project description:Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and progenitor cells, as well as in non-hematopoietic cells.

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Gene expression associated with lentiviral expression of PRAME in normal hematopoietic progenitors

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