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

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.

Fig. 1

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

Fig. 2

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

Fig. 3

Sensitivity and reproducibility of β-galactosidase assay. Different ratios of different shRNA plasmids and reporter fusion (pLC1) in cotransfection were employed. Concentration of shRNA plasmids for cotransfection was constant (5 μg) and pLC1 was 100 ng (light gray boxes), 500 ng (gray boxes), and 1 μg (dark gray boxes). Cells were analyzed 48 h after transfection

Fig. 4

Successful infection of B cell lymphoma cells (Granta 519) with lentivirus. a The efficiency of viral infection was analyzed, detecting GFP expression by FACS. Granta 519 control cells and infected cells were 65% viable 3 days after infection and infection ranged between 93.2% and 98.9%, dependent of MOI (15, 45, 90) used for transduction. b Specific knockdown effect for cyclin D1 was shown by Western blot from cell extracts prepared 3 days after infection with different MOI, for corresponding controls and cyclin D1 shRNA-Dh2. c qRT-PCR analysis of cyclin D1 mRNA was performed relative to the TBP housekeeping gene. Results are depicted as mRNA concentration relative to the control uninfected cells (Granta 519-c). Data were analyzed according to the ΔC_T method

Fig. 5

Successful infection of T cell lymphoma cells (Karpas 299 and SUDHL-1) with lentivirus. The efficiency of viral infection was analyzed, detecting GFP expression by FACS. a Karpas 299 noninfected and infected cells were 90% viable 3 days after infection and on average 75% infected. b SUDHL-1 control and infected cells were 43% viable 3 days after infection and 99% infected. c The level of knockdown effect for STAT3 was analyzed by Western blot from cell extracts prepared 3 days after infection with corresponding controls and STAT3 shRNA-Gh1. The specific downregulation of STAT3 protein was shown in the infected cells and no side effects were seen when membrane was probed with anti-ALK1. The same membrane was probed with anti-tubulin. d qRT-PCR analysis of STAT3 mRNA was performed relative to the TBP housekeeping gene. Results are depicted as mRNA concentration relative to Karpas 299 (light gray boxes) and SUDHL-1 (gray boxes) control cells. Data were analyzed according to the ΔC_T method