Rapid and highly efficient gene transfer into natural killer cells by nucleofection

Trompeter, Hans-Ingo; Weinhold, Sandra; Thiel, Corinna; Wernet, Peter; Uhrberg, Markus

doi:10.1016/s0022-1759(02)00431-3

Cited by 68 publications

(45 citation statements)

References 11 publications

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“…In order to do this, we needed to develop a method of achieving high levels of transfection in these primary cells. First, we investigated if we could transfect normal CD34 þ progenitors using the technique of modified electroporation known as nucleofection (Trompeter et al, 2003) using a GFP expressing plasmid. We found that normal CD34 þ cells rapidly express GFP following nucleofection with transfected cells detected within 3 h (Figure 2).…”

Section: Development Of a Technique For High-level Transient Transfecmentioning

confidence: 99%

“…This is a well-established technique in other cell types, in particular in colon carcinoma cell lines, but requires efficient transfection of a reporter construct, which is notoriously difficult in primary haemopoietic cells. We utilized the nucleofection technique, which has been reported to be effective in other primary cell types (Lai et al, 2003;Trompeter et al, 2003), and were able to achieve high levels of transient transfection in both CD34 þ cells and in AML blasts. This showed a low but detectable level of TCF/LEF reporter activity in normal CD34 þ cells.…”

Section: Cd34+ Patient Number --------------Aml Cases--------------mentioning

confidence: 99%

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Constitutive activation of the Wnt/β-catenin signalling pathway in acute myeloid leukaemia

et al. 2005

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The b-catenin protein is at the core of the canonical Wnt signalling pathway. Wnt stimulation leads to b-catenin accumulation, nuclear translocation and interaction with T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors to regulate genes important for embryonic development and proliferation. Wnt/b-catenin can promote stem cell self-renewal and is dysregulated in colon carcinoma. We have examined the role of the Wnt pathway in the development of acute myeloid leukaemia (AML) and find that the b-catenin protein is readily detected in primary AML samples. Using transfection of a TCF/LEF reporter construct into primary AML cells and normal human progenitors, we find increased reporter activity in 16/25 leukaemia samples. Retrovirally mediated expression of a mutant active b-catenin in normal progenitors preserves CD34 expression and impairs myelomonocytic differentiation. Activation of TCF/LEF signalling decreases factor withdrawal-induced apoptosis of normal progenitors. A significant proportion of AML cases show aberrant expression of components of the Wnt pathway including Wnt-1, Wnt-2b and LEF-1. These results provide evidence for the involvement of the Wnt/b-catenin pathway in the pathogenesis of AML.

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Section: Development Of a Technique For High-level Transient Transfecmentioning

confidence: 99%

Section: Cd34+ Patient Number --------------Aml Cases--------------mentioning

confidence: 99%

Constitutive activation of the Wnt/β-catenin signalling pathway in acute myeloid leukaemia

et al. 2005

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“…NK3.3 cells were transfected by nucleofection as described (39). Luciferase activity was analyzed 4 h after transfection by lysis of cells in 70 l of reporter lysis buffer followed immediately by luciferase activity measurement using the Luciferase Reporter Gene Assay (high sensitivity; Roche).…”

Section: Nucleofection Of Nk Cellsmentioning

confidence: 99%

“…Our use of the recently developed nucleofection technology enabled transfection efficiencies of NK cells of Ͼ50% and thus provided a suitable methodologic basis for highly sensitive functional studies of KIR promoters (39). Analysis initially focused on KIR2DL3 as a representative of other clonally distributed KIR genes with highly similar upstream regions.…”

Section: Expression Of Kir2dl3 As Well As Other Clonally Distributed mentioning

confidence: 99%

Three Structurally and Functionally Divergent Kinds of Promoters Regulate Expression of Clonally Distributed Killer Cell Ig-Like Receptors (KIR), of KIR2DL4, and of KIR3DL3

Trompeter

Gómez‐Lozano

Santourlidis

et al. 2005

The Journal of Immunology

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The generation of killer cell Ig-like receptor (KIR) expression patterns in NK cells involves variegated silencing of KIR genes by DNA methylation. To identify regulatory elements involved in KIR gene activation, upstream regions of KIR genes were functionally characterized in NK3.3 cells as well as in primary NK cells. Three kinds of KIR promoters were defined, controlling clonally expressed KIR genes, the constitutively active KIR2DL4, and the weakly expressed KIR3DL3. Upstream of a short core promoter common to all KIR genes, a region containing functionally divergent elements was characterized. Although this region had no impact on the activity of the KIR2DL3 promoter, an inhibitory element was identified in the KIR2DL4 promoter and an activating element was found in the KIR3DL3 promoter. Upon treatment with a methyltransferase inhibitor, KIR3DL3 expression could be readily induced showing that the low levels of KIR3DL3 expression in peripheral blood are due to sustained DNA methylation of an otherwise fully functional promoter. Analysis of transcription factor binding sites identified a functional acute myeloid leukemia (AML) site common to all three KIR promoters. Mutation of this site led to a substantial increase in activity of all KIR promoters. Among the different members of the AML family, AML-2 was identified as the predominant KIR binding factor. The present study suggests that AML-2 acts as a repressor of KIR expression in mature NK cells and opens the possibility that AML factors and associated cofactors are involved in regulation of KIR expression during NK cell development.

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“…Participation of NK cells in tumor lyses should induce local cytokine and chemokine secretion with the subsequent attraction of DCs, resulting in developing of memory cells and strong adaptive response. Development of a fast and simple technique of NK nucleofection 38 clears the way to the adaptation of the approach based on utilization of human NK cells modified with a chimeric immunoglobulin T-cell receptor. 39 After resolving the problem with the efficient NK cells genetic modification, this idea will be really attractive.…”

Section: Application Of Gene-targeted Effector Cells Like Lymphocytesmentioning

confidence: 99%

Gene transfer approaches in cancer immunotherapy

Larin

Georgiev

2004

Gene Ther

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Rapid and highly efficient gene transfer into natural killer cells by nucleofection

Cited by 68 publications

References 11 publications

Constitutive activation of the Wnt/β-catenin signalling pathway in acute myeloid leukaemia

Constitutive activation of the Wnt/β-catenin signalling pathway in acute myeloid leukaemia

Three Structurally and Functionally Divergent Kinds of Promoters Regulate Expression of Clonally Distributed Killer Cell Ig-Like Receptors (KIR), of KIR2DL4, and of KIR3DL3

Gene transfer approaches in cancer immunotherapy

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