Abstract
Different bacteria-derived systems for regulatable gene expression have been developed for the use in mammalian cells and some were also successfully adopted for in vivo use in vertebrate model organisms. However, certain limitations apply to most of these systems, including leakiness of transgene expression, inefficient transgene silencing or activation, as well as limited tissue accessibility of transgene-inducers or their unfavourable pharmacokinetics. In this study, we evaluated the suitability of the lac-operon/lac-repressor (lacO/lacI) system for the regulation of the well-established Vav-gene promoter that allows inducible transgene expression in different haematopoietic lineages in mice. Using the fluorescence marker protein Venus as a reporter, we observed that the lacO/lacI system could be amended to modulate transgene-expression in haematopoietic cells. However, reporter expression was not uniform and the lacO elements introduced into the Vav-gene promoter only conferred limited repression and reversion of lacI-mediated gene silencing after administration of IPTG. Although further optimization of the system is required, the lacO-modified version of the Vav-gene promoter may be adopted as a tool where low basal gene-expression and limited transient induction of protein expression are desired, e.g. for the activation of oncogenes or transgenes that act in a dominant-negative manner.
Highlights
Transgenesis in mice has become a useful tool to study gene function and model human diseases in vivo
A DNA element derived from the pOPI3CAT expression vector containing three symmetric lac operator binding sites recognized by the lac repressor, within a SV40-derived intron, was subcloned between the transcription start site of the endogenous Vav-gene and the multiple cloning site (MCS) of the HS21/45 VavPtransgenic vector
Venus encoding cDNA was subcloned into an unmodified version of the Vav-transgenic vector (VV) that served as a control
Summary
Transgenesis in mice has become a useful tool to study gene function and model human diseases in vivo. One of the problems connected with transgene overexpression is putative cytotoxicity, sometimes associated with induced lethality, but more frequently silencing of transgene expression and counter-selection of cells with low or no transgene expression Another limitation is related to the fact that expression of the target gene may be only desired in a specific cell type, at a specific developmental stage or for a limited time frame to better mimic events during normal development or human disease pathology. Well established in cell lines and today frequently used in transgenic mouse strains, certain limitations apply to these systems, mainly insufficient tightness of gene-repression and/or moderate induction levels, e.g., due to ineffective delivery and targeting of agonists to the cell type/tissue of interest, as well as stochastic epigenetic transgene silencing [4,5]
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