An in vivo transfection system for inducible gene expression and gene silencing in murine hepatocytes

Abstract

Our vector system combines Cre-lox mediated gene mutation with inducible gene expression or gene knockdown, respectively. It provides the opportunity for rapid and specific modification of hepatocyte gene expression and can be a useful tool for genetic screening approaches and analysis of target genes specifically in genetically engineered mouse models.

Constitutive and Inducible Systems for Genetic <em>In Vivo </em>Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

“…As transgenes or shRNA constructs are introduced by a fast and reliable recombinational cloning procedure, the system can easily be adapted for screening approaches. The vector system mediates reliable inducible expression in vivo that is entirely dependent on the delivery of doxycycline 11,30 . This practical video-based guide provides stepby-step instructions from cloning of suitable vectors over induction of gene expression to analysis of liver tissue.…”

“…This practical video-based guide provides stepby-step instructions from cloning of suitable vectors over induction of gene expression to analysis of liver tissue. However, to ensure efficiency of the system, several aspects should be kept in mind: To maintain long term expression, genomic integration is mediated at TA-sites by the sleeping beauty transposase 8,11 . Since integration efficiency is dependent on transposon size, it is important to keep the size of the transgene construct in mind when designing the vector 31 .…”

“…We previously generated a transposon construct that combines the constitutive expression of an inducible Cre recombinase (CreER) with inducible expression of a transgene or an shRNA of choice 20 (Figure 3A). Injection of this construct into Rosa26 mTmG -reporter mice and CreER activation by tamoxifen showed robust expression of the reporter gene comparable to the results obtained with a transposon construct for single transgene expression (Figure 3B).…”

Hydrodynamic Delivery: Characteristics, Applications, and Technological Advances

“…In terms of site-specific integration, PhiC31 integrase [197], sleeping beauty transposon [30,96,111,142,191,[199][200][201][202], piggyBac transposon [126,207], and Cre-loxP and technologies derived from it [208][209][210] were a major focus. Tamoxifen-dependent Cre recombinase (CreER) can initiate the recombination event at any desired time point [201,212]. In optogenetic genome engineering, split fragments of photoactivatable Cre can be hydrodynamically delivered and assembled so that the recombination process proceeds upon blue-light illumination, allowing for control of recombination events, not only in time but also in space [209].…”

Gene Therapy 2017: Progress and Future Directions

“…A number of promoter systems inducible by small molecule drugs have shown excellent dynamic range for gene regulation in cell culture and in animal models. Among these are the tetracycline inducible and repressible systems, and those based on modified estrogen and progesterone receptors . None of these has achieved clinical translation to date, primarily due to the immune responses to transcriptional activator domains that are not human in origin .…”