Molecular Biology and Genetics

Electronic Journal of Biotechnology ISSN: 0717-3458  
© 2006 by Pontificia Universidad Católica de Valparaíso -- Chile  
BIP RESEARCH ARTICLE

Multiple gene knock-down by a single lentiviral vector expressing an array of short hairpin RNAs 

Veronique Stove
Department of Clinical Chemistry
Microbiology and Immunology
Ghent University
Ghent University Hospital
4BlokA, De Pintelaan 185
9000 Ghent, Belgium
Tel: 32 9 2405871
Fax: 32 9 2404985
E-mail: Veronique.Stove@UGent.be 

Kaatje Smits
Department of Clinical Chemistry
Microbiology and Immunology
Ghent University
Ghent University Hospital
4BlokA, De Pintelaan 185
9000 Ghent, Belgium
Tel: 32 9 2405310
Fax: 32 9 2403659
E-mail: Kaatje.Smits@UGent.be

Evelien Naessens
Department of Clinical Chemistry
Microbiology and Immunology
Ghent University
Ghent University Hospital
4BlokA, De Pintelaan 185
9000 Ghent, Belgium
Tel: 32 9 2405310
Fax: 32 9 2403659
E-mail: Evelien.Naessens@UGent.be 

Jean Plum
Department of Clinical Chemistry
Microbiology and Immunology
Ghent University
Ghent University Hospital
4BlokA, De Pintelaan 185
9000 Ghent, Belgium
Tel: 32 9 2403641
Fax: 32 9 2403659
E-mail: Jean.Plum@UGent.be

Bruno Verhasselt*
Department of Clinical Chemistry
Microbiology and Immunology
Ghent University
Ghent University Hospital
4BlokA, De Pintelaan 185
9000 Ghent, Belgium
Tel: 32 9 2402226
Fax: 32 9 2404985
E-mail: bruno.verhasselt@UGent.be

*Corresponding author

Financial support: This work was supported by grants from the Research Foundation – Flanders (FWO). V.S. and K.S. equally contributed and are Ph.D. fellows of the FWO, and B.V. is a Senior Clinical Investigator of the FWO.

Keywords: EGFP, lentiviral gene transfer, multiple knock-down, RNAi, Rho GTPases, shRNA.

Abbreviations:

B2M: β2-microglobuline
CLTC: clathrin heavy chain
EGFP: enhanced green fluorescent protein
GTPases: guanosine triphosphatases
pol III: polymerase III
RNAi: RNA interference
SDF-1α: stromal cell-derived factor-1 alpha
shRNA: short hairpin RNA
siRNAs: small interfering RNAs

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RNA interference (RNAi) is a technique to block cellular protein expression, which became available around the turn of the millennium. RNAi is mediated by short double-stranded RNAs, and is a powerful mechanism for gene silencing due to specific degradation of protein encoding messenger RNA recognized by the short RNAs. For sustained intracellular expression, short hairpin RNA (shRNA) are an alternative to short double-stranded RNA as the latter have to reach the cell from the outer environment. Expression of shRNA can be accomplished in mammalian cells from plasmids or after viral delivery. Using lentiviral constructs (recombinant viruses derived from HIV-1), stable gene silencing is established both in dividing and non-dividing cells. However, targeting one single gene can lead to the development of escape mutants or may be insufficient to silence redundant pathways. Thus, for effective knock-down of signaling cascades or redundant protein pathways, several targets may have to be silenced in the same cell.

We further extended the use of lentiviral siRNA transgenesis by generating constructs expressing multiple shRNAs to achieve multiple protein knock-down. A RNAi cassette, consisting of  the H1 RNA pol III promoter and a gene-specific shRNA, is transferred to the widely used lentiviral vector pTRIP. This vector also encodes a green fluorescent reporter protein driven by a separate promoter. Green cells expressing EGFP are effectively transduced and therefore express the shRNA as well, enabling us to select cells with knockdown of a specific protein. Another RNAi cassette can be transferred to the same vector, thus creating tandem and in a next step triple constructs, making it possible to knock-down two or three genes with a single lentiviral vector. Our design allows an easy gradual extension of the number of RNAi cassettes.

We used this system to knock-down several members of the human Rho-family GTPases in T cells. Rho GTPases are a family of regulatory proteins, shown to play a role in different functions, such as cellular motility, adhesion and spatial organization of membrane receptors and signaling molecules. In hematopoietic cells like T cells, several Rho GTPases, e.g. Cdc42, Rac1 and Rac2 are expressed. These proteins are highly homologous and can have overlapping activity. Therefore, to study their function by knock-down, it might be necessary to target several of these small GTPases simultaneously.

Due to its consistent high Rac1, Rac2 and Cdc42 expression levels, the SupT1 T-cell line was chosen to verify the effectiveness of the shRNAs. Rac1, Rac2 and Cdc42 expression levels were assayed by Western blotting and real-time PCR. In cells transduced with single shRNA expressing virus, targeted protein expression was 6 to 17-fold less than in control transduced cells, while the other Rho GTPase proteins tested for were not affected. A similar ablation was seen in mRNA expression. In tandem and triple constructs, reduction in target gene expression was as strong as seen with single shRNA expressing viruses on protein level, although mRNA levels were less reduced. Similar experiments using the Jurkat T-cell line yielded the same results. These experiments showed that multiple shRNAs, expressed from the same lentiviral vector, are present at sufficient level to knock-down multiple targets. No apparent competition exists between multiple pol III promoters in close proximity and no saturation of the RNA-induced silencing complex RISC that destroys endogenous mRNAs complementary to the siRNA, was reached, as suggested previously.

As Rho GTPases are crucial proteins for directed cell migration, we performed transwell migration assays with SupT1 cells, known to respond chemotactically to stromal cell-derived factor-1 alpha (SDF-1α). Cells expressing shRNA specific for Rac1, Rac2 or Cdc42 migrated to a lower extent compared to cells not expressing shRNA, suggesting that targeted Rho GTPase proteins are all necessary for directional migration. Inhibition of migration correlated with EGFP levels, demonstrating that the marker gene expression, although driven by a separate promoter, is a reliable measure for shRNA levels. As expected from knock-down observed by immunoblot, cells transduced with virus encoding multiple shRNAs showed a disturbed migration. However, following elimination of two or three Rho GTPases, no additional effect on disturbance of chemotaxis was observed. Inhibition of chemotaxis was apparently complete with knock-down of either GTPase tested for, illustrated by similar behavior of tandem and triple compared to single shRNA transduced cells.

In conclusion, we present here a lentiviral shRNA delivery system that allows multiple shRNA species and a marker gene to be expressed from the same vector. The simultaneous knock-down of several targets within the same cell may be necessary, e.g. in compensatory or redundant pathways that have to be blocked at several levels, or to target hyper-variable viral sequences. Therefore, this strategy will likely prove to be of value in experimental and therapeutic applications.

Supported by UNESCO / MIRCEN network
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