Efficient shRNA delivery into B and T lymphoma cells using lentiviral vector-mediated transfer

被引：35

作者：

Anastasov N. ^{[1
,2
]}

Klier M. ^{[1
]}

Koch I. ^{[3
]}

Angermeier D. ^{[1
]}

Höfler H. ^{[1
,3
]}

Fend F. ^{[4
]}

Quintanilla-Martinez L. ^{[1
,5
]}

机构：

[1] Institute of Pathology, Helmholtz Center Munich, German Research Center for Environmental Health, Munich

[2] Institute of Radiation Biology, Helmholtz Center Munich, German Research Center for Environmental Health, Munich

[3] Institute of Pathology, Technical University Munich, Munich

[4] Institute of Pathology, Eberhard-Karls-University, Tuebingen

[5] University Hospital Tuebingen, Institute of Pathology, 72076 Tübingen

来源：

Journal of Hematopathology | 2009年 / 2卷 / 1期

关键词：

β-galactosidase assay; Anaplastic large cell lymphoma (ALCL); Efficient siRNA; Lentivirus transduction; Mantle cell lymphoma (MCL); RNA interference;

D O I：

10.1007/s12308-008-0020-x

中图分类号：

学科分类号：

摘要：

RNA interference is a powerful tool for the functional analysis of proteins by specific gene knockdown. In this study, we devised a rapid and efficient way to screen suitable siRNA sequences and subsequently employ them for specific gene knockdown in usually hard-to-transfect lymphoid cell lines, using a self-inactivating lentiviral vector. Two proteins with different half-lives were chosen, cyclin D1 and STAT3. A specific lacZ reporter fusion assay was used to identify highly effective siRNA sequences. Only siRNA molecules with more than 85% of knockdown efficiency were selected for the generation of lentiviral transfer vectors. Transduction rates of 75-99% were achieved in the lymphoma cell lines Granta 519 (mantle cell lymphoma), Karpas 299, and SUDHL-1 (anaplastic large T cell lymphoma), as demonstrated by green fluorescent protein expression in fluorescence-activated cell sorting analysis. The high level of transduction efficiency allows RNA interference studies to be performed on transduced cells without further manipulation, such as cell sorting or cloning. The LacZ reporter system together with the lentivirus technology is a very important tool in the hematology field, which enables experiments in lymphoid cells that were not possible before. © Springer-Verlag 2008.

引用

页码：9 / 19

页数：10

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