Expression of Transgenes Targeted to the Gt(ROSA)26Sor Locus Is Orientation Dependent

by Douglas Strathdee, Helen Ibbotson and Seth G. N. Grant

Abstract

Background

Targeting transgenes to a chosen location in the genome has a number of advantages. A single copy of the DNA construct can be inserted by targeting into regions of chromatin that allow the desired developmental and tissue-specific expression of the transgene.

Methodology

In order to develop a reliable system for reproducibly expressing trangenes it was decided to insert constructs at the Gt(ROSA)26Sor locus. A cytomegalovirus (CMV) promoter was used to drive expression of the Tetracycline (tet) transcriptional activator, rtTA2s-M2, and test the effectiveness of using the ROSA26 locus to allow transgene expression. The tet operator construct was inserted into one allele of ROSA26 and a tet responder construct controlling expression of EGFP was inserted into the other allele.

Conclusions

Expression of the targeted transgenes was shown to be affected by both the presence of selectable marker cassettes and by the orientation of the transgenes with respect to the endogenous ROSA26 promoter. These results suggest that transcriptional interference from the endogenous gene promoter or from promoters in the selectable marker cassettes may be affecting transgene expression at the locus. Additionally we have been able to determine the optimal orientation for transgene expression at the ROSA26 locus.

Citation: Strathdee D, Ibbotson H, Grant SGN (2006) Expression of Transgenes Targeted to the Gt(ROSA)26Sor Locus Is Orientation Dependent. PLoS ONE 1(1): e4. doi:10.1371/journal.pone.0000004

Academic Editor: Peter Fraser, The Babraham Institute, United Kingdom

Received: August 11, 2006; Accepted: August 31, 2006; Published: December 20, 2006

Copyright: © 2006 Strathdee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by a grant from the Gatsby Charitable Foundation and the Wellcome Trust.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Spatial and temporal control of gene expression represents an extremely powerful tool for the analysis of gene function and the events underlying complex biological processes such as embryonic development and cognitive function [1], [2].

One method of reversibly controlling gene activity is to regulate its transcription [3]. The transcription control system based on elements of the tetracycline (tet) resistance operon of E. coli have been widely used to control gene expression in mammalian cells. One of the key components of the tet system is the tetracycline-controlled transactivator (tTA), a fusion protein between the repressor of the Tn10 tet resistance operon of Escherichia coli and a C-terminal portion of VP16 that contains domains capable of activating transcription [4]. tTA will activate transcription from a suitably engineered minimal promoter by binding to an array of tet operator sequences positioned upstream. Random mutagenesis of TetR generated a new transactivator (rtTA), which binds and transactivates gene expression in the presence of doxycycline (dox) [5]. Improved versions of rtTA have been developed to give tighter gene expression, increased sensitivity towards the inducer, enhanced stability and expression in mammalian cells, and more uniform transgene expression in the induced cells [6]–[8].

Introduced transgenes are frequently susceptible to gene silencing [9]. Although the mechanism for this process is poorly understood, both the integration site and the variable copy number at the integration site can influence the expression level [9]–[11]. This is often seen as progressive silencing of gene expression initially resulting in mosaic expression levels and often resulting in complete shutdown of transgene expression [12].

As randomly integrated transgenes are susceptible to gene silencing, targeting transgenes to a chosen location in the mouse genome has a number of advantages [13], [14]. Firstly the integration site can be chosen to allow insertion of the transgene into a region of chromatin favourable for expression and that avoids an undesirable insertional mutagenesis. Additionally only a single copy is be introduced which avoids problems associated with a large multicopy array.

The Gt(ROSA)26Sor locus (ROSA26) was first described as a gene trap which is ubiquitously expressed in mouse embryos [15]. As the ROSA26 locus is active in most cells any promoter inserted into the locus should not be restricted in its expression by unfavourable chromatin configurations. This locus has been widely used for expressing endogenous sequences, often reporter genes usually from the endogenous promoter [16], [17]. The promoter from the ROSA26 locus has been used to drive widespread expression of marker genes in transgenic rats and mice [18]. The ROSA26 promoter has also been used to express the tTA and rtTA successfully [19]–[21]. Other studies have shown that targeting tissue-specific promoters, including BAC sequences, to specific genomic locations leads to its expression in the appropriate tissue and cell-specific pattern [22]–[26].

In order to test if the ROSA26 locus was and if the local chromatin structure would effect transgene expression at the locus constructs expressing the rtTA and a reporter gene downstream of the tetracycline-responsive element (TRE) were targeted into ROSA26 locus in both orientations. Expression was found to be dependent on both the orientation of the transgene and the presence of an adjacent selectable marker.

Results

Targeting strategy for introducing to ROSA26 locus

Establishing dox-dependent gene expression requires two different transgenes, an activator and a responder transgene. Activator constructs were generated with a CMV promoter driving the rtTA2s-M2 variant of the tetracycline transcriptional activator. The CMV promoter was chosen as it works inefficiently in ES cells and should be sensitive to position effects [27]. The cassette was cloned into the targeting vector in both orientations to produce the A1 and A2 targeting constructs (Figure 1A). The responder construct contained an EGFP transgene under control of the tetracycline responder element. This again was cloned into the targeting vector in both orientations to produce the B1 and B2 targeting vectors.