Efficient gene transfer in CLL by mRNA electroporation

Abstract

Chronic lymphocytic leukemia (CLL) consists of at least two major prognostic subgroups, characterized by different cellular and molecular markers. This observation sparked studies on the function and clinical importance of these markers. In order to address their function adequately, an efficient and reliable method for gene transfer is needed. In this study, we compared efficiency and utility of different gene transfer techniques in CLL. Lenti-, retro-and adenoviral transduction did not yield appreciable numbers of marker gene enhanced green fluorescent protein (EGFP) positive CLL cells, despite various prestimulation protocols. Efficient transgene expression was observed after nucleofection of CLL cells with plasmid DNA, at the expense of low survival rates. After optimization, electroporation of in vitro transcribed mRNA yielded up to 90% EGFP þ CLL cells without affecting survival. Transgene expression remained detectable for at least 2 weeks after electroporation. Furthermore, we could demonstrate overexpression of ZAP70 and of a ZAP70-EGFP fusion protein after electroporation with ZAP70 or ZAP70-EGFP mRNA. We conclude that mRNA electroporation is a novel and straightforward method for highly efficient gene transfer in CLL. The application of this technique should facilitate functional studies on CLL cells, as well as clinical research.

Disruption of autophagy by the histone deacetylase inhibitor MGCD0103 and its therapeutic implication in B-cell chronic lymphocytic leukemia

“…To confirm this finding, siRNA-mediated knockdown of key autophagy genes was performed. In agreement with previous reports, 28, 29 primary CLL cells were highly refractory to transfections, probably owing to their quiescent nature. Nevertheless, in three out of seven CLL samples analyzed, introduction of either ATG5 , MAP1LC3B , BECN1 or ATG5/BECN1 siRNAs resulted in decreased target gene expression (ranging from 22 to 40% when compared with cells treated with scrambled siRNAs), together with decreased cell viability (Figures 6b–d).…”

CLL Cells Respond to B-Cell Receptor Stimulation with a MicroRNA/mRNA Signature Associated with MYC Activation and Cell Cycle Progression

“…Cells were cultured as described before [20]. BCR stimulation was performed as described by Kofler et al [21] Anti-IgM-polyacrylamid immunobead (anti-IgM) reagent (Irvine Scientific, Santa Ana, CA, USA) was added to the PBMC cultures at a concentration of 100 µg/mL for 3 or 24 hours.…”

Histone deacetylases mediate the silencing of miR-15a, miR-16, and miR-29b in chronic lymphocytic leukemia

“…All miRs were transfected at 300nM using the Amaxa nucleofector and the cell line nucleofector kit V, program U13 35 into primary leukemia cells (5 ϫ 10 6 /nucleofection) for 48 hours, after which cells were harvested for RNA and protein analysis. In standardization experiments, the transfection efficiency ranged between 41% and 50% based on the uptake of the fluorescent transfection indicator siGLO (Dharmacon) (data not shown).…”