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

Anastasov N, Klier M, Koch I, Angermeier D, Höfler H, Fend F, Quintanilla-Martinez L - J Hematop (2008)

Bottom Line: A specific lacZ reporter fusion assay was used to identify highly effective siRNA sequences.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

shRNAs against cyclin D1 are able to specifically silence lacZ-cyclin D1 reporter expression and endogenously expressed cyclin D1. a Cotransfection of different shRNA constructs for cyclin D1 with lacZ-cyclin D1 reporter fusion (pLC1). shRNA effect was measured 48 h after transfection by β-galactosidase assay. pSuper and pS control represent controls with empty vector and pSuper with control shRNA sequence, respectively. STAT3 shRNA-Gh1 was used as an irrelevant control for lacZ-cyclin D1 reporter expression. b Western blot for cyclin D1 demonstrates striking reduction of both endogenous cyclin D1 as well as fusion protein in the cells cotransfected with specific shRNA constructs (pS-Dh2 and pS-SA1) 48 h after transfection. The same membrane was reprobed with anti-tubulin
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Fig2: shRNAs against cyclin D1 are able to specifically silence lacZ-cyclin D1 reporter expression and endogenously expressed cyclin D1. a Cotransfection of different shRNA constructs for cyclin D1 with lacZ-cyclin D1 reporter fusion (pLC1). shRNA effect was measured 48 h after transfection by β-galactosidase assay. pSuper and pS control represent controls with empty vector and pSuper with control shRNA sequence, respectively. STAT3 shRNA-Gh1 was used as an irrelevant control for lacZ-cyclin D1 reporter expression. b Western blot for cyclin D1 demonstrates striking reduction of both endogenous cyclin D1 as well as fusion protein in the cells cotransfected with specific shRNA constructs (pS-Dh2 and pS-SA1) 48 h after transfection. The same membrane was reprobed with anti-tubulin

Mentions: To precisely quantify the shRNA effect on cyclin D1, specific fusion protein lacZ-cyclin D1 (pLC1, described in “Materials and methods”) was constructed and transfected into the 293-T-cell line. Cotransfection of pSuper (pS) empty vector or control (pSS) shRNA sequence with pLC1 did not change the expression of the fusion protein significantly. Constructs pS-Dh1, pS-Dh3, and pS-SA1 were characterized as moderately active molecules with a knockdown efficiency of 80%. Construct pS-Dh2 was characterized as highly active shRNA with 96% of knockdown efficiency. The other shRNAs failed to show significant activity. Construct pS-Gh1, with the best knockdown efficiency for STAT3, was used as an independent control for specificity of the lacZ-cyclin D1 fusion knockdown. Cotransfection of STAT3 shRNA-Gh1 with fusion plasmid (pLC1) showed no effect on lacZ-cyclin D1 expression in the 293-T-cell line (Fig. 2a).Fig. 2


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

Anastasov N, Klier M, Koch I, Angermeier D, Höfler H, Fend F, Quintanilla-Martinez L - J Hematop (2008)

shRNAs against cyclin D1 are able to specifically silence lacZ-cyclin D1 reporter expression and endogenously expressed cyclin D1. a Cotransfection of different shRNA constructs for cyclin D1 with lacZ-cyclin D1 reporter fusion (pLC1). shRNA effect was measured 48 h after transfection by β-galactosidase assay. pSuper and pS control represent controls with empty vector and pSuper with control shRNA sequence, respectively. STAT3 shRNA-Gh1 was used as an irrelevant control for lacZ-cyclin D1 reporter expression. b Western blot for cyclin D1 demonstrates striking reduction of both endogenous cyclin D1 as well as fusion protein in the cells cotransfected with specific shRNA constructs (pS-Dh2 and pS-SA1) 48 h after transfection. The same membrane was reprobed with anti-tubulin
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Related In: Results  -  Collection

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Fig2: shRNAs against cyclin D1 are able to specifically silence lacZ-cyclin D1 reporter expression and endogenously expressed cyclin D1. a Cotransfection of different shRNA constructs for cyclin D1 with lacZ-cyclin D1 reporter fusion (pLC1). shRNA effect was measured 48 h after transfection by β-galactosidase assay. pSuper and pS control represent controls with empty vector and pSuper with control shRNA sequence, respectively. STAT3 shRNA-Gh1 was used as an irrelevant control for lacZ-cyclin D1 reporter expression. b Western blot for cyclin D1 demonstrates striking reduction of both endogenous cyclin D1 as well as fusion protein in the cells cotransfected with specific shRNA constructs (pS-Dh2 and pS-SA1) 48 h after transfection. The same membrane was reprobed with anti-tubulin
Mentions: To precisely quantify the shRNA effect on cyclin D1, specific fusion protein lacZ-cyclin D1 (pLC1, described in “Materials and methods”) was constructed and transfected into the 293-T-cell line. Cotransfection of pSuper (pS) empty vector or control (pSS) shRNA sequence with pLC1 did not change the expression of the fusion protein significantly. Constructs pS-Dh1, pS-Dh3, and pS-SA1 were characterized as moderately active molecules with a knockdown efficiency of 80%. Construct pS-Dh2 was characterized as highly active shRNA with 96% of knockdown efficiency. The other shRNAs failed to show significant activity. Construct pS-Gh1, with the best knockdown efficiency for STAT3, was used as an independent control for specificity of the lacZ-cyclin D1 fusion knockdown. Cotransfection of STAT3 shRNA-Gh1 with fusion plasmid (pLC1) showed no effect on lacZ-cyclin D1 expression in the 293-T-cell line (Fig. 2a).Fig. 2

Bottom Line: A specific lacZ reporter fusion assay was used to identify highly effective siRNA sequences.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany.

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.

No MeSH data available.


Related in: MedlinePlus