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

Journal of hematopathology (2009-08-12)
Natasa Anastasov, Margit Klier, Ina Koch, Daniela Angermeier, Heinz Höfler, Falko Fend, Leticia Quintanilla-Martinez
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ampicillin, Ready Made Solution, 100 mg/mL, 0.2 μm filtered
Sigma-Aldrich
Ampicillin, anhydrous, 96.0-102.0% (anhydrous basis)
Sigma-Aldrich
LB Agar Ampicillin-100, Plates, pre-poured LB agar plates with 100 μg/ml ampicillin
Sigma-Aldrich
Ampicillin, meets USP testing specifications

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