Heritable and inducible gene knockdown in astrocytes or neurons in vivo by a combined lentiviral and RNAi approach

Abstract

Gene knockout by homologous recombination is a popular method to study gene functions in the mouse in vivo. However, its lack of temporal control has limited the interpretation of knockout studies because the complete elimination of a gene product often alters developmental processes, and can induce severe malformations or lethality. Conditional gene knockdown has emerged as a compelling alternative to gene knockout, an approach well-established in vitro but that remains challenging in vivo, especially in the adult brain. Here, we report a method for conditional and cell-specific gene knockdown in the mouse brain in vivo that combines Cre-mediated RNA interference (RNAi) with classical and lentivirus-mediated transgenesis. The method is based on the inducible expression of a silencing short hairpin RNA (shRNA) introduced in mice by lentivirus-mediated transgenesis, and on its activation by excision of a floxed stop EGFP reporter with an inducible Cre recombinase expressed in astrocytes or in neurons. This dual system should be of broad utility for comparative studies of gene functions in these two cell types in vivo. Keywords: in vivo gene knockdown, conditional gene knockdown, shRNA, Cre-lox genetics, lentivirus-mediated transgenesis INTRODUCTIONGenetic models in the mouse have significantly advanced the understanding of gene functions, both in physiological and pathological conditions. Most of the existing models have been generated by genetic manipulations that lead to a gain-or loss-offunction of the candidate gene (Aronoff and Petersen, 2006). This is usually achieved with vector-based overexpression systems, or by gene targeting like gene knockout or knockin (Capecchi, 2005;Glaser et al., 2005). Recently, RNA interference (RNAi) has emerged as an alternative approach with the advantage of decreasing rather than fully eliminating the expression of target gene(s) Paddison and Hannon, 2002). RNAi-based gene knockdown relies on short doublestranded RNAs (19-21 nucleotides) that target complementary mRNA sequences, and promote their degradation or inhibit their translation Dykxhoorn et al., 2003). It can be used in rodents by local or systemic delivery of shortinterfering RNAs (siRNAs) (Thakker et al., 2004), or through virus-based shRNA expression vectors (Ralph et al., 2005;Singer et al., 2005;Sapru et al., 2006). Although this approach can be extremely useful, its efficiency is subjected to variability and depends on several factors, in particular the degree of sequence matching with the target DNA, and the level of expression of the shRNA and the targeted gene. Further, even if it has been used successfully in cell culture (Elbashir et al., 2001;Brummelkamp et al., 2002;Lee et al., 2002), it remains challenging in vivo. Thus, overall, genetic systems for gene silencing have been reported in the literature (Rubinson et al., 2003;Tiscornia et al., 2003;Coumoul et al., 2005;Szulc et al., 2006;Dickins et al., 2007;Delic et al., 2008;Seidler et al., 2008) but are often difficult to use due to their limitations s...

Cre Recombinase and Other Tyrosine Recombinases

“…One problem for RNAi from expressed constructs in cells is that the short hairpins are typically expressed from constitutive Poll III promoters. T-SSRs have helped to make the method conditional and reversible to allow inducible hairpin expression in cells, , mice, , and plants. , The system has also been adapted for use employing lentiviral vectors, providing improved delivery into difficult to transfect cells and in a therapeutic setting to combat hepatocellular carcinoma in a mouse model . Furthermore, the system has been extended to turn alleles on or off using Cre recombinase to rapidly address questions of tissue-specific and cell autonomy of gene function in development …”

Lentiviral vectors as tools to understand central nervous system biology in mammalian model organisms

“…Conditional knockdown of gene expression is another powerful method to analyze protein function in the adult brain. microRNA (miRNA)-based short hairpin knockdown can be combined with cell specific promoters ( Nielsen et al, 2009 ), or used in combination with cre-lox or drug-inducible cre-lox systems for spatial and temporal control of gene knockdown ( Stern et al, 2008 ; Heitz et al, 2014 ). For example, Heitz et al (2014) use a lentiviral vector expressing a floxed GFP miRNA to transduce mice expressing GFAP or CaMKII regulated tamoxifen-inducible cre (creERT2).…”

Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System

“…Recently, Ventura and his colleagues developed pSico vector for stable expression of shRNA, with conditional excision of shRNA cassette by cre recombinase [ 19 ]. This system has been widely utilized for cell-type-specific gene-silencing [ 19 20 21 22 ]. However, pSico has a critical limitation that gene-silenced cells cannot be identified by fluorescence, due to the excision of the reporter gene.…”