Regulation of Endothelial-Specific Transgene Expression by the LacI Repressor Protein In Vivo

Abstract

Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2) with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacIR, and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacIR protein.

Inducible and reversible silencing of the Pvalb gene in mice: An in vitro and in vivo study

“…Six founders (four for shPV22 and two for shTurboGFP, see Table ) were subsequently used to establish transgenic lines by breeding them with PV‐Cre mice. Three lines (#277 and #284 for shPV22; #236 for shTurboGFP) were further bred to PV‐Cre mice until having offspring of the third generation (F3), as transgene inheritance is generally unstable in early generations (Morton, Chaston, Baillie, Hill, & Matthaei, ). The identification of mice carrying the transgene in these generations was based on the visualization of eGFP + cells (using the Incucyte Imaging system) from peritoneal aspirates.…”

“…IPTG is well tolerated also after long exposure with no noticeable side effects (Cronin et al., ). IPTG is taken up rapidly by all tissues including the endothelium (Morton et al., ) and brain tissue, as it crosses the blood–brain barrier and is highly stable intracellularly (Wyborski & Short, ). As with all systemic application methods (oral, i.p.…”

“…IPTG is well tolerated also after long exposure with no noticeable side effects (Cronin et al, 2001). IPTG is taken up rapidly by all tissues including the endothelium (Morton et al, 2014) and brain tissue, as it crosses the blood-brain barrier and is highly stable intracellularly (Wyborski & Short, 1991). As with F I G U R E 5 PV immunofluorescence staining of cortex (left panel), reticular thalamic nucleus (RTN) and striatum (right panel) of an untreated (-IPTG) shPV22 mouse (line #277), a shPV22 transgenic mouse treated with IPTG (experimental conditions as shown in Figure 4) all systemic application methods (oral, i.p.…”

Peptide Uptake Is Essential for Borrelia burgdorferi Viability and Involves Structural and Regulatory Complexity of its Oligopeptide Transporter

“…To assess the morphology of peptide-starved mammalian-host-adapted spirochetes, DMCs were implanted with unwashed NBD mutant spirochetes at 1 × 10 5 spirochetes/ml and incubated for 1 week. Three days prior to implantation, rats were given water alone or IPTG-treated water (2% sucrose solution containing 80 mM IPTG) ( 93 ) and were then maintained under the same condition for the remainder of the experiment.…”

Generation of reporter hESCs by targeting EGFP at the CD144 locus to facilitate the endothelial differentiation

“…Given that the promoterless EGFP could be driven by the endogenous EC-specific promoter, in the present study we selected the CD144 locus, one of the classic markers of ECs (Lampugnani et al, 1992;Mao et al, 2013;Morton, Chaston, Baillie, Hill, & Matthaei, 2014), as the target site for tracing endothelial differentiation.…”