High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34+ cells via electroporation-mediated gene delivery

Phosphorylation and O-Linked Glycosylation of Elf-1 Leads to Its Translocation to the Nucleus and Binding to the Promoter of the TCR ζ-Chain

“…We first transfected primary human T and Jurkat cells, using a cell electroporation technique (11)(12)(13)(14), either with the plasmid-encoding cDNA (pcDNA)/Elf-1 plasmid, which encodes the full-length Elf-1 cDNA, or with the empty plasmid to establish the protein products of the Elf-1 gene. The cytoplasmic extracts from primary ( Fig.…”

“…The electroporation methods used for the transfection of Jurkat and primary T lymphocytes recently have been described (11)(12)(13)(14).…”

Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element

“…Primary human fibroblast-like cells, pEPI-eGFP is detected in semisolid colonies derived from transfected CD34 + cells Next, we transfected CD34 + -enriched cells isolated from umbilical cord blood using electroporation. [28][29][30][31][32] Transfection efficiency was between 10 and 30% (mean: 18.478%, n ¼ 5), as estimated by flow cytometric evaluation of the percentage of eGFP-expressing CD34 + cells. Cell survival was in all cases above 60% (mean: 66.876%, n ¼ 5).…”

Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation

“…[6][7][8][9][10] Although cell synchronization and electroporation studies have led to controversial conclusions on the requirement for S-phase, [11][12][13] it appears that efficient electroporation of hematopoietic cells requires prestimulation, which results in an apparent increase in the S-phase sub-population. 6,14,15 Ideal culture conditions would promote a high efficiency of gene transfer to ensure not only short-term, but also long-term engraftment and subsequent bone marrow repopulation with each hematopoietic lineage carrying the transgene. In vitro manipulation of human stem cells for gene transfer by electroporation involves three primary goals: (1) optimization of short-term culture conditions that will increase the percentage of cells in S-phase and enhance successful incorporation of exogenous gene delivered; (2) maintenance of the pluripotent stem cells for full bone marrow engraftment and long-term reconstitution after transplant; and (3) optimization of the electroporation conditions for efficient transfection.…”