Silencing gene expression by RNA interference (RNAi) can provide insight into gene function but requires efficient delivery of small interfering RNAs (siRNAs) into cells. Introduction of exogenous nucleic acids can be especially difficult in cultured pancreatic islets. This article describes a method for making recombinant adenoviruses that efficiently drive expression of siRNAs in islet β-cells and a β-cell–derived cell line. Transduction with a virus expressing an siRNA specific for GLUT2 reduced GLUT2 mRNA and protein levels by 80% in the INS-1–derived β-cell line, 832/13, and GLUT2 protein levels by >90% in primary rat islets. Another virus expressing an siRNA specific for glucokinase (GK) caused 80% suppression of GK mRNA and 50% suppression of GK protein levels in 832/13 cells. These experiments validate recombinant adenoviral RNAi vectors as a useful tool for suppression of the expression of specific genes in pancreatic islets and β-cell lines. Advantages of this approach include 1) the high efficiency of adenovirus-mediated gene transfer in insulinoma cell lines and rat islets and 2) the rapidity with which RNAi constructs can be prepared and tested relative to stable-transfection strategies.
Loss of pancreatic islet β-cell function and mass occurs in both major forms of diabetes. Better therapies for these diseases could therefore emanate from a clearer understanding of genes that control key aspects of β-cell biology. To this end, several laboratories have used expression-profiling methods to define changes in gene expression that occur in models of β-cell differentiation (1–3) and responses to stress (4,5). However, such studies often yield long lists of differentially expressed genes, constituting a new bottleneck for understanding of the molecular basis of the phenotype under study.
RNA interference (RNAi), recently recognized as a specific and efficient method for gene silencing in cells, uses intracellular short interfering RNAs (siRNAs) to mediate destruction of the cognate mRNA species (6–8). Initial investigations of RNAi in mammalian cells relied on transfection with synthetic siRNAs (8) or plasmids designed to drive expression of siRNAs through the use of RNA polymerase III promoters (9). Unfortunately, transfection efficiency is often low when oligonucleotides or plasmids are given to nondividing primary cells. This is especially true of cultured pancreatic islets, which exist as spheroidal clusters of 100–2,000 cells that are difficult to transfect by physical methods. We have previously demonstrated the utility of recombinant adenoviruses for high-efficiency gene transfer into isolated pancreatic islets in culture (10–14). Here, we report the design, construction, and testing of an adenoviral shuttle vector that is used as part of an established adenoviral platform (10) to generate siRNA-expressing adenoviruses. We show that these viruses mediate highly efficient suppression of target genes in pancreatic islets and in an insulinoma cell line.
RESEARCH DESIGN AND METHODS
Relative to the start codon, the 5′ ends of the targets correspond to rat GLUT2 (accession number J03145) nucleotide 15 (GAT CAC CGG AAC CTT GGC T) and rat islet glucokinase (GK) (accession number M25807) nucleotide 202 (GGC TCA GAA GTC GGA GAC T). A target in the Photinus pyralis luciferase gene, GL2 (8), was used as a negative control. For each target, sense and antisense oligonucleotides were designed and synthesized, as described (9). Oligonucleotides were annealed in salt/Tris/EDTA buffer (10 mmol/l Tris, 1 mmol/l EDTA, 50 mmol/l NaCl, pH 8.0) and ligated into BglII/HindIII-linearized pSUPER (9; OligoEngine, Seattle, WA).
An adenoviral shuttle vector, EH006, was prepared by replacing the 1.2-kbp cytomegalovirus (CMV) promoter–based NotI expression cassette from pAC.CMV (10,15) with a mini-cloning cassette, constructed by annealing oligonucleotides TB468 (GGC CAT GGA TCC GAA TTC GTT TAA ACG AAG CTT TA) and TB469 (GGC CTA AAG CTT CGT TTA AAC GAA TTC GGA TCC AT) in STE.
For each pSUPER-based clone, the siRNA expression cassette was excised with EcoRI-HindIII and ligated into EcoRI-HindIII–linearized EH006 (Fig. 1A). To create RNAi adenoviruses by homologous recombination, EH006-derived shuttle vectors were cotransfected with the adenoviral plasmid, pJM17 (10), into human embryonic kidney cells (HEK293; American Type Culture Collection, Manassas, VA). Infectious titers of tertiary viral lysates, determined by end point dilution assay in HEK293 cells (16), ranged from 2.0 × 109 to 1.0 × 1010 plaque-forming units (pfu)/ml.
Gene silencing in transgenic insulinoma cells.
The rat insulinoma line 832/13 was cultured as described (17). Cells in late log growth were plated at 52,600 cells/cm2. One day later, the medium was removed and cells were transduced at viral doses of up to 240 pfu/cell for 2 h. Virus was removed, and fresh medium was added. At 24-h intervals, cells were lysed, RNA was isolated, and GLUT2 and GK transcript levels were assayed by real-time PCR analysis, using prevalidated primer and probes (Applied Biosystems). Three days after viral treatment, GK and GLUT2 protein levels were also evaluated by immunoblot analysis using polyclonal anti-GLUT2 (46701-659; Biogenesis, Poole, U.K.) and GK (GCK; Santa Cruz Biotechnology, Santa Cruz, CA) antisera and a horseradish peroxidase–conjugated, anti-rabbit secondary antibody (Amersham Biosciences, Little Chalfont, U.K.). Sample loading was monitored by measurement of total protein (Bio-Rad) and staining of gels with Coomassie Blue after protein transfer (not shown).
Gene silencing in rat islets of Langerhans.
Under a protocol approved by the Duke University Institutional Animal Care and Use Committee, pancreatic islets of Langerhans were isolated from male Wistar rats, 200–250 g, as described (18). Culture medium was that of Hohmeier et al. (17), with glucose adjusted to 7.2 mmol/l. Islets in aliquots of 360 per well of a six-well plate in 2 ml medium were transduced with recombinant RNAi adenoviruses for 20 h at a dose of 1.9 × 106 pfu/islet. After transduction, islets were cultured for 3 days before measurement of GLUT2 protein levels by immunoblot, as described above.
Construction of siRNA-expressing adenoviruses.
We constructed an adenoviral shuttle vector, EH006 (Fig. 1A), that is designed to accept siRNA expression cassettes from the siRNA expression vector pSUPER (9). Importantly, EH006 was derived from the widely used adenoviral shuttle vector pAC.CMV (10,15) by replacement of the CMV-based expression cassette with a mini-cloning cassette, making this vector accessible via long-established and widely used methods and reagents. Recombinant viruses are created by cotransfection of HEK293 cells with an EH006-derived shuttle vector and pJM17 (10,15), a replication-defective variant of the adenoviral genome. After in vivo recombination, a recombinant viral genome is generated as shown in Fig. 1B. These viruses are predicted to express a short RNA hairpin (Fig. 1C), which is further processed to the bioactive siRNA by removal of the loop sequence by the mammalian homolog of the RNase III nuclease, Dicer.
Adenoviral-mediated silencing in insulinoma cells.
To test the ability of recombinant adenoviruses to direct RNAi-mediated gene silencing, we assayed GLUT2 and GK expression in an INS-1 insulinoma cell line, 832/13, after transduction with the Ad-GLUT2-siRNA and Ad-GK-siRNA viruses, respectively.
Treatment of 832/13 cells with the Ad-GLUT2-siRNA and Ad-GK-siRNA viruses caused decreases in GLUT2 and GK transcript levels in as little as 24 h after transduction, with maximal suppression of 70–80% observed 3 days after transduction (Fig. 2A). Compared with levels in untreated cells, we noted that GLUT2 and GK levels in Ad-luc-siRNA–transduced cells were slightly reduced 2 days after transduction, but, in both cases, GLUT2 and GK levels recovered by 3 days after transduction.
Treatment of 832/13 cells with Ad-GLUT2-siRNA reduced GLUT2 protein levels by 80% (Fig. 2B), whereas treatment with Ad-GK-siRNA reduced GK protein levels by 50% (Fig. 2C). Treatment of the same cells with a control virus (Ad-luc-siRNA) had no effect on GLUT2 or GK protein levels. Because GLUT2 and GK RNA levels were reduced to a similar extent after transduction, the lesser efficiency of suppression of GK protein levels is likely attributable to differences in rates of protein turnover.
Adenoviral-mediated RNAi silenced GLUT2 expression in cultured rat islets.
Although important insights can be gained by genetic engineering studies in insulinoma cell lines, key concepts must always be confirmed in primary cell preparations. We therefore tested the utility of our new adenovirus vectors for delivery of siRNAs into cultured rat islets. As shown in Fig. 3, treatment of cultured islets with 1.9 × 106 pfu/islet of Ad-GLUT2-siRNA resulted in a >95% reduction in GLUT2 expression compared with untreated islets and a >90% reduction when compared with Ad-luc-siRNA–treated islets.
Pancreatic islets of Langerhans play a critical role in control of metabolic fuel homeostasis. Islet function and mass are lost or disturbed in diabetes, and this has stimulated research efforts focused on identification of genes that can enhance islet function and survival. Multiple reports have appeared on microarray analysis for identification of genes involved in regulation of islet cell development (1), insulin secretion (2,3), and stress responses (4,5) in β-cell model systems. However, functional studies that prove the involvement of specific genes in defining the phenotype under study have been lacking. This report describes a vector system that should be useful for defining the “functional genomics” of the β-cell, since it allows rapid analysis of candidate genes in tractable in vitro systems, including primary preparations of pancreatic islets. Although a number of researchers have previously described adenoviral-based RNAi systems (19,20), the current study describes an adenoviral RNAi shuttle vector that is based on a well-established adenoviral platform (10,15) already in widespread use among diabetes researchers (21–24) and is the first to describe siRNA-mediated gene suppression in primary islet cells. Moreover, the suppression of one target gene (GLUT2) by more than 90% in primary cultures of rat islets strongly supports the utility of our methods for gene silencing within β-cells, since these cells constitute ∼70–80% of the total islet cell population and are the main site of islet GLUT2 expression.
A number of other RNAi viral systems have also been previously described, including retroviral (25) and lentiviral (26,27) systems. The genomes of these viruses integrate into the host genome, leading to long-term expression, in contrast to the adenoviral genome, which remains episomal, resulting in relatively transient expression. However, recombinant adenoviruses can be grown to much higher titers than lenti- and retroviruses, and this characteristic may make adenoviruses particularly useful tools for RNAi-mediated gene silencing, since intracellular viral copy number and therefore expression of siRNAs may be significantly higher. Adenoviral reagents that drive overexpression of selected genes have proven themselves useful in metabolic research into diabetes and obesity, both in vitro and in vivo (10–14,28). Data in this report demonstrate that islets and insulinoma cell lines can be targeted for selective gene suppression using recombinant RNAi adenoviruses.
J.R.B. and J.C.S. contributed equally to this work.
This work was supported by the National Institutes of Health (NIH) (2-U01-DK-56047-04) and by a sponsored research agreement with Takeda Chemicals. The vectors described in this manuscript were developed with funding from an NIH β-cell consortium program grant. The vectors are intended for broad use in the β-cell research community, and any investigators interested in obtaining the vectors should contact Dr. Becker at firstname.lastname@example.org.
Hans-Ewald Hohmeier, Danhong Lu, Mette Valentin-Jensen, and Helena Winfield provided valuable insight and technical assistance.