Development of a BAC vector for integration-independent and tight regulation of transgenes in rodents via the Tet system

Abstract

The establishment of functional transgenic mouse lines is often limited by problems caused by integration site effects on the expression construct. Similarly, tetracycline (Tet) controlled transcription units most commonly used for conditional transgene expression in mice are strongly influenced by their genomic surrounding. Using bacterial artificial chromosome (BAC) technology in constitutive expression systems, it has been shown that integration site effects resulting in unwanted expression patterns can be largely eliminated. Here we describe a strategy to minimize unfavourable integration effects on conditional expression constructs based on a 75 kb genomic BAC fragment. This fragment was derived from a transgenic mouse line, termed LC-1, which carries the Tet-inducible genes luciferase and cre (Schönig et al. 2002). Animals of this mouse line have previously been shown to exhibit optimal expression properties in terms of tightness in the off state and the absolute level of induction, when mated to appropriate transactivator expressing mice. Here we report the cloning and identification of the transgenic LC-1 integration site which was subsequently inserted into a bacterial artificial chromosome. We demonstrate that this vector facilitates the efficient generation of transgenic mouse and rat lines, where the Tet-controlled expression unit is shielded from perturbations caused by the integration site.

Conditional gene expression systems in the transgenic rat brain

“…The experimental strategy for creating the EGFP-Ptetbi-luc lines has previously been described [22]. In brief, a 75 kb genomic mouse fragment containing the Ptetbi-controlled transcription unit was used for microinjection into fertilized SD rat oocytes.…”

“…In brief, a 75 kb genomic mouse fragment containing the Ptetbi-controlled transcription unit was used for microinjection into fertilized SD rat oocytes. In mice, these genomic sequences have been shown to minimize the influence of the genomic integration site on tet-regulated promoters [22,23]. Two transgenic founders harbouring the full-length transgene could be identified.…”

Tetracycline Inducible Gene Manipulation in Serotonergic Neurons

“…This holds true for any randomly integrated transgene but is particularly worrisome in an inducible system which requires two transgenes to optimally work together. Position effects can be minimized by using large genomic sequences to drive transactivator or Ptet-controlled gene expression [42], [44]. For that reason, we used a large genomic Tph2 sequence to insulate tTA expression from position effects.…”

2015 Guidelines for Establishing Genetically Modified Rat Models for Cardiovascular Research

“…Plasmid-based transgenes and bacterial artificial chromosome (BAC)-based transgenes are routinely used in rats [10, 67–69] and BACs that are recombineered to carry Cre recombinase transgenes have recently been used to produce some of the first Cre transgenic models in rats [65, 68, 70]. Lentivirus-based transgenesis is another established technique in the rat [71, 72], although this transgenesis method is considerably less prevalent.…”