Identification of internal ribosome entry site (IRES) activity

Description

We provide a technological platform to investigate putative IRES activity use various assay systems to determine the precise location of the IRES element in the gene cluster. Once identified, a functional IRES element can be used to develop an IRES-based multiple gene expression vector for the efficient co-expression of multiple genes. These vectors have several important applications. For example, they can be used for gene therapy; they can be designed to coordinate the expression of functionally related proteins in order to simultaneously deliver multiple defense genes; or they can be used to express a single gene with a marker.

Brief protocol

Plasmid construction

The pRL-FL plasmid was constructed as described previously (Bieleski and Talbot, 2001), that is, the firefly luciferase from pGL3 plasmid (Promega) was inserted into pRL-null plasmid (Promega) to give the dual luciferase plasmid T7/pR-F. For transient DNA transfection in Sf9 cells, the WSSV ie1 promoter (−94/+52) (Liu et al., 2007) was used to replace the T7 promoter by amplification and cloning the promoter into the SacI-NheI sites of T7/pR-F to produce the construct ie1/pR-F. The putative IRES fragment and its antisense sequence (as a negative control) were PCR amplified and cloned into ie1/pR-F to generate ie1/pR-IRES-F. For in vitro translation, we used the plasmids T7/pR-IRES-F. These were identical to ie1/pR-IRES-F except that they used the original T7 promoter.

In vitro transcription

RNA transcripts were prepared using a RiboMAX™Large Scale RNA Production System-T7 kit (Promega) as described by the technical manual. The various T7/pR-F based constructs were linearized with NotI, and T7 RNA polymerase (Promega) was used to transcribe uncapped transcripts from the linearized plasmid DNAs (1 μg) at 37 °C for 2 h. The DNA template was then removed by digestion using RNase-free DNase I (Invitrogen) as described above. RNA sizes were determined by separating the RNAs on 1% agarose-formaldehyde gel.

In vitro translation

In vitro translation of the uncapped transcripts was performed by Flexi Rabbit Reticulocyte Lysate (RRL; Promega) according to the manufacturer's instructions. RNA (1.5 μg) was added to 25 μl reaction mixtures containing 16.5 μl of RRL, 1 μl of [35S] methionine, 0.5 μl of amino acid mixture minus methionine, 0.5 μl of RNasin® ribonuclease inhibitor (40 U/μl; Promega), 0.5 μl of 100mMDTT, and 0.4 μl of 2.5M potassium chloride (final potassium ion concentration was 40 mM) and incubated at 30 °C for 90 min. An aliquot of the reaction products (5 μl) was then added to 5 μl of 2× SDS sample buffer and analyzed by SDS-PAGE and autography as described above.

IRES activity assay for transfected Sf9 cells

For the IRES activity assays, Sf9 cells were seeded in 24-well trays (1×10⁵ cells/well) and grown in Sf-900 II SFM serum-free medium (Invitrogen) overnight at 27 °C. Plasmid DNAs (0.5 μg of plasmid DNA per well) were transfected into Sf9 cells using the Cellfectin reagent (Invitrogen) according to the manufacturer's recommendations. Cells were harvested at 48 h after transfection and analyzed for dual luciferase activities using the Dual-Luciferase® Reporter Assay System (Promega). Briefly, transfected cells were washed twice with 1× PBS, lysed with 100 μl of passive lysis buffer, and then incubated for 15min at RT on an orbital shaker with gentle shaking. Cell lysate (20 μl)were used to measure luciferase activities with a Labsystems benchtop luminometer. The ratio of firefly luciferase activity to Renilla luciferase activity was used as an index of IRES activity. Transfection assays were performed in triplicate with three independent experiments, each using a different batch of purified plasmid DNA. Data are presented as mean±SD (standard derivation) from the three triplicate experiments.

References

1. Kang, S. T., J. H. Leu, et al. (2009). "Polycistronic mRNAs and internal ribosome entry site elements (IRES) are widely used by white spot syndrome virus (WSSV) structural protein genes." Virology 387(2): 353-363.

Some of our publications that have used this platform

1. Kang, S. T., J. H. Leu, et al. (2009). "Polycistronic mRNAs and internal ribosome entry site elements (IRES) are widely used by white spot syndrome virus (WSSV) structural protein genes." Virology 387(2): 353-363.