Limits...
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

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 ΔCT method
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2713496&req=5

Fig4: 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 ΔCT method

Mentions: Since lentiviruses can infect a wide range of dividing and nondividing cells, we tested whether the lentiviral vector pFUGW can be used for siRNA-mediated gene silencing in human lymphoma cell lines. The most efficient construct in β-galactosidase assay for cyclin D1 (Fig. 3), pS-Dh2, was used for lentivirus transfer vector production. The resulting lentiviral vector pF-Dh2 was packaged and first titrated on 293 T cells for determination of MOI. To identify the titer required for efficient transduction, the mantle cell lymphoma cell line Granta 519 was transduced with increasing MOI (15, 45, and 90; Fig. 4a–c). Although the viability (65%) and the transduction efficiency was equally good with the different MOI, the amount of shRNA in the cells was increasingly higher with 45 and 90 MOI, as judged by the intensity of GFP in the FACS analysis (Fig. 4a). Western blot analysis showed an excellent correlation between cyclin D1 protein expression and the amount of MOI used, with practically complete knockdown of cyclin D1 with 90 MOI (Fig. 4b). However, quantitative RT-PCR of cyclin D1 mRNA demonstrated similar cyclin D1 levels between 45 and 90 MOI, whereas, with 90 MOI nonspecific downregulation of cyclin D1 mRNA was observed in the controls (Fig. 4c). Therefore, 40–45 MOI were used for all subsequent experiments in lymphoma cell lines. In Granta 519 cells and corresponding controls, high levels (98% on average) of GFP were expressed 3 days after transduction, with specific cyclin D1 downregulation as shown in Fig. 4b.Fig. 4


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)

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 ΔCT method
© Copyright Policy
Related In: Results  -  Collection

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

Fig4: 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 ΔCT method
Mentions: Since lentiviruses can infect a wide range of dividing and nondividing cells, we tested whether the lentiviral vector pFUGW can be used for siRNA-mediated gene silencing in human lymphoma cell lines. The most efficient construct in β-galactosidase assay for cyclin D1 (Fig. 3), pS-Dh2, was used for lentivirus transfer vector production. The resulting lentiviral vector pF-Dh2 was packaged and first titrated on 293 T cells for determination of MOI. To identify the titer required for efficient transduction, the mantle cell lymphoma cell line Granta 519 was transduced with increasing MOI (15, 45, and 90; Fig. 4a–c). Although the viability (65%) and the transduction efficiency was equally good with the different MOI, the amount of shRNA in the cells was increasingly higher with 45 and 90 MOI, as judged by the intensity of GFP in the FACS analysis (Fig. 4a). Western blot analysis showed an excellent correlation between cyclin D1 protein expression and the amount of MOI used, with practically complete knockdown of cyclin D1 with 90 MOI (Fig. 4b). However, quantitative RT-PCR of cyclin D1 mRNA demonstrated similar cyclin D1 levels between 45 and 90 MOI, whereas, with 90 MOI nonspecific downregulation of cyclin D1 mRNA was observed in the controls (Fig. 4c). Therefore, 40–45 MOI were used for all subsequent experiments in lymphoma cell lines. In Granta 519 cells and corresponding controls, high levels (98% on average) of GFP were expressed 3 days after transduction, with specific cyclin D1 downregulation as shown in Fig. 4b.Fig. 4

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