Delivery of Short Hairpin RNA Sequences by Using a Replication-Competent Avian Retroviral Vector

While recent studies have demonstrated that retroviral vectorscan be used to stably express short hairpin RNA (shRNA) to inhibitgene expression, these studies have utilized replication-defectiveretroviruses. We describe the creation of a replication-competent,Gateway-compatible retroviral vector capable of expressing shRNAthat inhibits the expression of specific genes.

INTRODUCTION

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Introduction

Retroviral vectors can be used to express short hairpin RNA(shRNA) under the control of an RNA polymerase III (Pol III)promoter, such as U6 or H1, for the purpose of inhibiting geneexpression in a sequence-specific manner (3, 9, 15). However,all studies to date have utilized replication-defective retrovirusesthat require packaging cell lines or helper virus for virusproduction. A series of replication-competent retroviral vectorshave been developed based on the Schmitt Ruppin A (SR-A) strainof Rous sarcoma virus (reviewed in reference 6). These vectors,referred to as RCASBP(A) or RCANBP(A), have several advantagesover other retroviral vectors: (i) in avian cells, these vectorsare replication competent; (ii) they routinely achieve hightiters in avian cells (10⁷ infectious units/ml); and (iii) inmammalian cells, infectious viruses are not produced, allowingfor multiple rounds of infection into the same cell while preventinguncontrolled cell-to-cell spread (5, 7). Furthermore, successfulinfection of mammalian cells by the RCASBP/RCANBP(A) virus requiresthe use of vectors with non-avian envelope proteins, such asthe amphotropic envelope from murine leukemia virus (1), orectopic expression of the avian retroviral receptor TVA on thecell surface (2, 19). For this study, we used cells expressingTVA to allow cell-specific targeted infection of mammalian cells(5). We demonstrate that RCANBP(A)-mediated delivery of shRNAtargeted against glyceraldehyde-3-phosphate dehydrogenase (GAPDH)can specifically reduce GAPDH expression in mammalian cells.

Development of a Gateway-compatible RCANBP(A) viral vector.

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Development of a gateway...

We developed a replication-competent retroviral vector expressingshRNA under the control of the human H1 promoter (Fig. 1). Thisvector was engineered to be Gateway compatible to facilitateefficient cloning of DNA sequences into the viral vector (constructiondetails available upon request). The Gateway-compatible RCANBP(A)vector (Fig. 1A) was used to generate RCANBP-H1-GAPDH, whichcontains an RNA Pol III-shRNA cassette consisting of the humanH1 promoter upstream of a GAPDH shRNA hairpin-loop sequence(Ambion, Austin, Tex.) (Fig. 1B). This viral vector producedreplication-competent virus in the immortalized chicken fibroblastcell line DF-1 (16), as determined by enzyme-linked immunosorbentassay (17), and had a high titer relative to RCASBP(A)AP, whichwas used as a standard (data not shown).

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FIG. 1. Schematic representation of a Gateway-compatible, replication-competent, retroviral vector capable of delivering shRNA. (A) Gateway-compatible RCANBP(A). (B) The RCANBP-H1-shRNA viral vector has been engineered to express shRNA by introducing an RNA Pol III-shRNA cassette, containing the H1 promoter upstream of the shRNA stem-loop sequence. SA, splice acceptor; SD, splice donor; LTR, long terminal repeat; ${psi}$ , packaging signal.

Generation of TVA-positive cell lines.

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Generation of tva-positive cell...

A stable clonal line, A375-TVA, from the human malignant melanomacell line A375 (8) was created by transfection of a pCDNA3.1/hygroconstruct (Invitrogen, Carlsbad, Calif.) containing the TVAgene and selection in medium containing 300-µg/ml hygromycinB. The percentage of TVA-positive cells in this clonal linewas assessed by infection with RCASBP(A)-GFP virus (12). FACSanalysis indicated that more than 90% of the cells were infectedby RCASBP(A)-GFP (data not shown). These data indicate thatthe A375-TVA cell line is competent for RCASBP/RCANBP(A) infection.

RNAi of GAPDH expression in RCANBP-H1-GAPDH-infected cells.

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Rnai of gapdh expression...

A375-TVA cells were infected over 2 days with the RCANBP-H1-GAPDHvirus in the presence of 8-µg/ml Polybrene (Sigma, St.Louis, Mo.), and RNA was extracted after one or two rounds ofinfection. Polyacrylamide gel electrophoresis-Northern analysisshowed that both the one- and two-round-infected A375-TVA cellsproduced antisense GAPDH RNA, and this RNA was not seen in uninfectedcells (Fig. 2A). Comparatively, our retroviral system appearsto be more efficient at expressing shRNA than the other retroviralsystems, as the RCANBP-H1-GAPDH virus produced 100 to 300 fmolof shRNA per µg of cellular RNA analyzed (Fig. 2A), whereasother retroviral vectors express at most 3 fmol of shRNA perµg of cellular RNA (18). Northern analysis of GAPDH messagein A375-TVA cells infected with RCANBP-H1-GAPDH virus showedthat the GAPDH message was drastically reduced after only oneround of infection (Fig. 2B), and this effect was maintainedover four rounds of infection. This reduction in GAPDH messagelevels is not apparent on day 5 (data not shown). Analysis ofcell proliferation over five rounds of RCANBP-H1-GAPDH infectionindicated that there were fewer RCANBP-H1-GAPDH-infected cellsrelative to uninfected cells. However, cell number began torecover after 5 days, and by 13 days, it had recovered to thelevel seen in the uninfected cells (data not shown). GAPDH isan important ATP-generating enzyme in glycolysis, and a decreasein activity leads to ATP depletion and cell death (4). The initiallag in cell number may be due to cell death associated withthe loss of GAPDH expression. Recovery of both cell number andGAPDH message after 5 days in RCANBP-H1-GAPDH-infected A375-TVAcells was associated with loss of TVA-positive cells in culture(data not shown). Infection of A375-TVA cells with a virus containingan unrelated shRNA had no effect on cell growth (data not shown).

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FIG. 2. Ability of RCANBP-H1-GAPDH to express GAPDH shRNA and to reduce GAPDH expression in A375-TVA cells. (A) Polyacrylamide gel electrophoresis-Northern analysis of GAPDH shRNA expression using the sense strand as an RNA probe. A375-TVA cells were uninfected (– [control]) or were infected with RCANBP-H1-GAPDH for one (1x) or two (2x) rounds. Samples were normalized for total RNA content, and 70 µg of total cellular RNA was analyzed per sample. On the sense strand, GAPDH shRNA (19 nucleotides [nt]) was used at 100, 10, and 1 pmol as a size marker and for quantitation of shRNA production. The upper band likely corresponds to hairpin-loop formation (47 nt) of the shRNA prior to loop digestion. (B) Northern blot analysis of GAPDH message in A375-TVA cells that were uninfected (lane 1) or infected with RCANBP-H1-GAPDH (lane 2) for one round. Blots were stripped and reprobed with 18S RNA to ensure equal loading of RNA.

In this report, we describe the development of a replication-competentretroviral vector, RCANBP-H1-GAPDH, containing shRNA sequencesspecific for human GAPDH under the control of the human H1 promoter.This vector was replication competent in DF-1 cells and expressedGAPDH shRNA after infection of the human melanoma cell line,A375 (Fig. 2A), which expressed the viral receptor TVA. Infectionof A375-TVA cells resulted in reduction of GAPDH message afteronly one round of infection. These data indicate that the RCANBP(A)/TVAsystem can be used for RNA interference (RNAi)-based silencingof specific genes in avian and mammalian cells in vitro. Thisretroviral vector takes advantage of the tissue- and cell-specifictargeted infection by the RCANBP(A)/TVA system and avoids thedisadvantages of replication-defective retroviruses. RNAi studiesare rapid, cost-effective, and can be easily adapted for studiesin numerous organisms. This delivery system should greatly enhancethe utility of RNAi-based studies in both avian and mammaliansystems in vivo and in cell culture. RCASBP/RCANBP-based retroviralvectors have been used to study viral replication, avian development,and cancer in mice (5, 7, 10, 11, 13, 14). The majority of thesestudies have analyzed gain-of-function phenotypes by deliveringand overexpressing the gene(s) of interest. This study extendsthe utility of the RCANBP(A)/TVA system to include loss-of-functionanalyses of specific genes in vitro as well as in vivo.

ACKNOWLEDGMENTS

We thank Stacie Loftus for advice and Eric Holland for the SPKEQ8TVAconstruct. We thank Stephen Hughes for advice, for providingthe RCANBP(A) vector, and for critical review of the manuscript.

This work was supported by the Van Andel Research Institute.S. L. Holmen is a Pfizer Fellow of the Life Sciences ResearchFoundation.

FOOTNOTES

* Corresponding author. Mailing address: Van Andel Research Institute, 333 Bostwick NE, Grand Rapids, MI 49503. Phone: (616) 234-5308. Fax: (616) 234-5309. E-mail: bart.williams{at}vai.org.

REFERENCES

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