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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 STAT3 specifically silence lacZ-STAT3 reporter expression as well as endogenously expressed STAT3 in a 293-T-cell cotransfection model. a Simultaneous delivery of different STAT3 shRNAs with the lacZ-STAT3 fusion plasmid induces reduction of β-galactosidase activity. β-galactosidase activity is normalized to 100% in 293 T cells transfected only with specific LacZ fusion. pSuper represents empty vector control. Cells were analyzed 48 h after transfection. b Western blot for STAT3 protein of cells transfected with lacZ-STAT3 reporter (pLS3) and simultaneously with control plasmid (pSuper) or different shRNA plasmids (pS-Gh1, pS-Inv1, pS-Dh353, and pS-Dh17) 48 h and c 72 h after transfection. In contrast to the fusion protein, endogenous STAT3 disappears only at 72 h after transfection. The membranes were reprobed with antitubulin to confirm equal loading
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Fig1: shRNAs against STAT3 specifically silence lacZ-STAT3 reporter expression as well as endogenously expressed STAT3 in a 293-T-cell cotransfection model. a Simultaneous delivery of different STAT3 shRNAs with the lacZ-STAT3 fusion plasmid induces reduction of β-galactosidase activity. β-galactosidase activity is normalized to 100% in 293 T cells transfected only with specific LacZ fusion. pSuper represents empty vector control. Cells were analyzed 48 h after transfection. b Western blot for STAT3 protein of cells transfected with lacZ-STAT3 reporter (pLS3) and simultaneously with control plasmid (pSuper) or different shRNA plasmids (pS-Gh1, pS-Inv1, pS-Dh353, and pS-Dh17) 48 h and c 72 h after transfection. In contrast to the fusion protein, endogenous STAT3 disappears only at 72 h after transfection. The membranes were reprobed with antitubulin to confirm equal loading

Mentions: Specific fusion protein lacZ-STAT3 (pLS3) was first constructed (described in “Materials and methods”) to precisely quantify the shRNA effect on STAT3 expression. The β-galactosidase activity was normalized to 100% with the activity of the fusion protein in cell extracts from 293 T cells transfected only with lacZ-STAT3 fusion (pLS3; Fig. 1a). The knockdown efficiency of four STAT3 shRNA constructs was investigated. All four designed sequences for STAT3 knockdown were effective on expression of lacZ-STAT3 fusion (Fig. 1a), showing striking reduction in β-galactosidase activity. The best knockdown effect was achieved with the constructs pS-Gh1 (97%) and pS-Dh353 (98%), whereas the other two constructs pS-INV1 and pS-Dh17 showed moderate activity with 80% and 75% knockdown efficiency, respectively. In order to see whether the effect was specific, cotransfection of cyclin D1 shRNA–pSuper expression vector (pS-Dh2) with pLS3 was performed. pS-Dh2 showed induction of lacZ-STAT3 fusion activity up to 195%, possibly due to the presence of a negative feedback loop between cyclin D1 and STAT3.Fig. 1


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 STAT3 specifically silence lacZ-STAT3 reporter expression as well as endogenously expressed STAT3 in a 293-T-cell cotransfection model. a Simultaneous delivery of different STAT3 shRNAs with the lacZ-STAT3 fusion plasmid induces reduction of β-galactosidase activity. β-galactosidase activity is normalized to 100% in 293 T cells transfected only with specific LacZ fusion. pSuper represents empty vector control. Cells were analyzed 48 h after transfection. b Western blot for STAT3 protein of cells transfected with lacZ-STAT3 reporter (pLS3) and simultaneously with control plasmid (pSuper) or different shRNA plasmids (pS-Gh1, pS-Inv1, pS-Dh353, and pS-Dh17) 48 h and c 72 h after transfection. In contrast to the fusion protein, endogenous STAT3 disappears only at 72 h after transfection. The membranes were reprobed with antitubulin to confirm equal loading
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713496&req=5

Fig1: shRNAs against STAT3 specifically silence lacZ-STAT3 reporter expression as well as endogenously expressed STAT3 in a 293-T-cell cotransfection model. a Simultaneous delivery of different STAT3 shRNAs with the lacZ-STAT3 fusion plasmid induces reduction of β-galactosidase activity. β-galactosidase activity is normalized to 100% in 293 T cells transfected only with specific LacZ fusion. pSuper represents empty vector control. Cells were analyzed 48 h after transfection. b Western blot for STAT3 protein of cells transfected with lacZ-STAT3 reporter (pLS3) and simultaneously with control plasmid (pSuper) or different shRNA plasmids (pS-Gh1, pS-Inv1, pS-Dh353, and pS-Dh17) 48 h and c 72 h after transfection. In contrast to the fusion protein, endogenous STAT3 disappears only at 72 h after transfection. The membranes were reprobed with antitubulin to confirm equal loading
Mentions: Specific fusion protein lacZ-STAT3 (pLS3) was first constructed (described in “Materials and methods”) to precisely quantify the shRNA effect on STAT3 expression. The β-galactosidase activity was normalized to 100% with the activity of the fusion protein in cell extracts from 293 T cells transfected only with lacZ-STAT3 fusion (pLS3; Fig. 1a). The knockdown efficiency of four STAT3 shRNA constructs was investigated. All four designed sequences for STAT3 knockdown were effective on expression of lacZ-STAT3 fusion (Fig. 1a), showing striking reduction in β-galactosidase activity. The best knockdown effect was achieved with the constructs pS-Gh1 (97%) and pS-Dh353 (98%), whereas the other two constructs pS-INV1 and pS-Dh17 showed moderate activity with 80% and 75% knockdown efficiency, respectively. In order to see whether the effect was specific, cotransfection of cyclin D1 shRNA–pSuper expression vector (pS-Dh2) with pLS3 was performed. pS-Dh2 showed induction of lacZ-STAT3 fusion activity up to 195%, possibly due to the presence of a negative feedback loop between cyclin D1 and STAT3.Fig. 1

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