Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats

Abstract

Currently, tools to generate loss-of-function mutations in rats are limited. Therefore, we have developed a lentiviral single-vector system for the temporal control of ubiquitous shRNA expression. Here, we report transgenic rats carrying an insulin receptor-specific shRNA transcribed from a regulatable promoter and identified by concomitant EGFP expression. In the absence of the inducer doxycycline (Dox), we observed no siRNA expression. However, Dox treatment at very low concentrations led to a rapid induction of the siRNA and ablation of INSR protein expression. As anticipated, blood glucose levels increased, whereas insulin signaling and glucose regulation were impaired. Importantly, this phenotype was reversible (i.e., discontinuation of Dox treatment led to INSR re-expression and remission of diabetes symptoms). The lentiviral system offers a simple tool for reversible gene ablation in the rat and can be used for other species that cannot be manipulated by conventional recombination techniques.diabetes ͉ transgenesis ͉ RNAi G ene targeting by homologous recombination in embryonic stem cells is a powerful tool to generate loss-of-function mutations in mice (1). Nevertheless, additional strategies to inhibit gene expression are desirable because this technique is not applicable to other species, is time-and cost-intensive, and is not reversible (2). RNA interference (RNAi) holds great promise as an experimental tool to achieve this goal. The method is based on the introduction of siRNAs into cells. After binding to the target transcript, the resulting dsRNA complex is incorporated into RNA-induced silencing complex (RISC), finally leading to mRNA degradation and inhibition of translation (3, 4). Experimentally, siRNAs can be introduced into cells by expressing a shRNA under the control of RNA polymerase III promoters, including H1 (5, 6). Incorporation of the shRNA cassette into viral vectors allows for inheritable gene knockdown in cells and even whole animals (7-9).Lentiviruses have the unique ability to infect nondividing cells. Therefore, they have been used for the generation of transgenic models in various species, including overexpression and genesilencing studies in mice (7-9) and rats (10-12). Temporal inactivation of genes employing inducible systems for the expression of shRNAs have been developed in vitro (13,14). These systems are based on the expression of silencing RNAs by RNA polymerase III promoters containing operator sequences (tetO) of the Escherichia coli tetracycline resistance (tet) operon. Recently, a mouse model for tight control of RNAi was established (15), exploiting the reversible inhibition of gene transcription following binding of doxycycline (Dox) to the tet repressor (tetR). Using a similar approach, we developed a lentiviral single-vector system containing an H1-tetO-shRNA cassette and the codon-optimized tetR linked to EGFP.Insulin resistance is a hallmark of type 2 diabetes mellitus, one of the most prevalent metabolic diseases of the Western world (16, 17). Impaired insul...

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

“…The Tet-Off/Tet-On lentiviral vector approach can also be used for inducible knockdown of gene expression in vivo, by expressing short hairpin RNA under the control of an inducible promoter, as shown in rats with inducible ablation of the insulin-receptor expression. 43 Finally, the study of human-specific pathogenesis can also take advantage of conditional-live pathogens, in which replication is depending on the Tet-On gene regulation system. Recently, we have generated a conditional-live HIV-1 variant in which life cycle is dependent on the Tet-On gene regulation system (HIVrtTA).…”

Stable silencing of the glucocorticoid receptor in myelin‐specific T effector cells by retroviral delivery of shRNA: Insight into neuroinflammatory disease

“…Second, the knockout technology is limited to mice since homologous recombination in ES cells is not yet available in other species. Therefore, the advantage of certain rat models of brain autoimmune diseases [28] can only be exploited by generating knockdown animals [10,11] or by AT of cells after RNAi-mediated gene silencing. Third, the generation of knockout mice is costly and time-consuming, for which reason it does not qualify for largescale screenings.…”

“…Experimentally, stable introduction of siRNA into cells can be achieved by expressing a small hairpin RNA (shRNA) using retroviral or lentiviral vectors. By this means, the state of silencing is inherited over many cell divisions or animal generations [10,11] and allows for detailed analyses of gene function in vivo.…”

Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM

“…Cells were transfected using the calcium phosphate precipitation method as described previously. 38 The generation of the Cas9 expression vector and sgRNA constructs was described recently. 20 The sgRNA sequences are as follows:…”