Inducible vav cre

• Megumi Takiguchi, 
• Lukas E. Dow, 
• Julia E. Prier, 
• Catherine L. Carmichael, 
• Benjamin T. Kile, 
• Stephen J. Turner, 
• Scott W. Lowe, 
• David C. S. Huang, 
• Ross A. Dickins

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Abstract

Citation: Takiguchi M, Dow LE, Prier JE, Carmichael CL, Kile BT, Turner SJ, et al. (2013) Variability of Inducible Expression across the Hematopoietic System of Tetracycline Transactivator Transgenic Mice. PLoS ONE 8(1): e54009. https://doi.org/10.1371/journal.pone.0054009

Editor: Nathalie Labrecque, Maisonneuve-Rosemont Hospital, Canada

Received: August 7, 2012; Accepted: December 6, 2012; Published: January 11, 2013

Copyright: © 2013 Takiguchi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, inducible vav cre permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are best vav This work was supported by the National Health and Medical Research Council of Australia (Project grants 575535 and 1024599 and Inducible vav cre 509693 to RAD), Australian Government National Health and Medical Research Council Independent Research Institutes Infrastructure Support Scheme, the Sylvia and Charles Viertel Charitable Foundation (Fellowships to BTK and RAD), Victorian State Government OIS grants, Australian Research Council Future Fellowship (awarded to SJT) and the Victorian Endowment for Science, Knowledge and Innovation (VESKI Fellowship to RAD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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

Introduction

Genetically modified mice are important tools for the study of mammalian gene function in vivo. Targeted gene modification using homologous recombination in mouse embryonic stem cells allows production of mice with mutations in specific genes, and the Cre/lox system allows conditional deletion of genes in particular cell types [1]–[3]. The tetracycline (tet)-regulated expression system is also widely used in mouse models, with the key advantage of temporal and reversible control of transgene expression. Originally developed for inducible gene overexpression [4]–[6], it also allows inducible gene knockdown via expression of microRNA-based inducible vav cre hairpin RNAs (shRNAs) [7]–[9].

The tet-regulated system requires two genetic components: a tet response element (TRE) promoter controlling mRNA or shRNA expression, and a recombinant tet-transactivator that can activate the TRE promoter. The tTA (tet-off) transactivator binds and activates the TRE promoter but is inhibited by the administration of tetracycline or its commonly used analog doxycycline (Dox). Conversely, the rtTA (tet-on) transactivator is latent until activated by Dox. Therefore Dox indirectly controls expression from the TRE by reversibly regulating transactivator function.

In vivo tet-regulated protein or shRNA expression is commonly achieved by crossing mice carrying a TRE promoter cassette transgene with mice carrying a tet transactivator transgene, resulting in progeny carrying both genetic components. An important factor in effective tet-regulated expression is the genomic location of the TRE promoter cassette, which influences its accessibility by the tet transactivator. Hence, vav puerto rico approaches have targeted the TRE cassette to defined genomic loci to optimise inducible expression in inducible vav cre cell types [9], [10]. A second key determinant of effective tet-regulation is the expression level of the tet transactivator. Many mouse strains have been generated that express the tTA or rtTA transactivators under the control of different promoters (www.tetsystems.com). Although many of these promoters are nominally ubiquitous or tissue-specific, in most cases the pattern and abundance of inducible vav cre expression in these mouse strains is poorly characterised. In order to optimally utilise transgenic, tet-regulated expression systems in mice, and to rationally interpret the resulting phenotypes, an understanding of the strength and breadth of transactivator function in particular cell types in vivo inducible vav cre imperative. In this study we have examined in vivo transactivator function across the hematopoietic system of several commonly used transactivator mouse strains.

Results

Characterising Tet-regulated Expression in Hematopoietic Stem and Progenitor Cells

To examine tet-regulated expression in the hematopoietic system of transgenic transactivator mouse strains, we utilised a reporter inducible vav cre strain where expression of green fluorescent protein (GFP) is under the control of the TRE promoter. The 3′ UTR of the GFP-encoding transcript in this reporter strain also includes a microRNA-based shRNA targeting firefly luciferase (Luc.1309 or shLuc) [9]. We have previously used this TRE-GFP-shLuc strain as a negative control in tet-regulated shRNA studies [9], [11]. The TRE-GFP-shLuc transgene is targeted to the collagen type I alpha (Col1a1) locus, previously shown to facilitate tet-regulated expression in a wide range of cell types in vivo[9], [10]. GFP expression was negligible in hematopoietic cells of TRE-GFP-shLuc inducible vav cre transgenic mice, verifying that reporter expression is not leaky (Figure S1).

We crossed reporter mice to various transgenic transactivator mouse strains procured or produced by our laboratories. More recently developed tet-on mouse strains often express the M2-rtTA or rtTA3 transactivators, which have improved transcriptional activity and Dox-sensitivity relative to the original rtTA protein [12], [13]. Of the six transgenic transactivator mouse strains we examined, four express the tet-on transactivator: CAG-rtTA3 [9], CMV-rtTA [14], ROSA26-M2rtTA [15], and Vav-rtTA3. The CAG, CMV, and ROSA26 promoters are often regarded as ‘ubiquitous’ promoters and are widely used to drive a broad expression pattern in transgenic mice. The CAG promoter contains an enhancer element from human cytomegalovirus (CMV) together with sequences from the chicken ß-actin promoter and rabbit ß-globin genes that yield high level expression in mammalian cells [16]. Similarly, the CMV promoter is inducible vav cre on the strong promoter of the immediate early gene of human CMV [17]. The ROSA26 promoter was originally identified based on its broad expression pattern during mouse embryogenesis but is also widely active in adult tissues [18], [19]. In ROSA26-M2rtTA mice M2rtTA expression is driven by the endogenous ROSA26 promoter, whereas the other five strains tested were all originally generated by pronuclear injection of synthetic expression cassettes resulting in variable transgene insertion site and copy number. To complement the broadly acting tet-on mouse strains CAG-rtTA3, CMV-rtTA, and ROSA26-M2rtTA, we inducible vav cre standard pronuclear transgenesis to generate a transgenic mouse strain where expression of the rtTA3 transactivator is under control of the Vav promoter. Vav promoter activity is inducible vav cre restricted to the hematopoietic compartment of mice, where it drives expression across all blood cell types [20]. After screening several independent transgenic founder lines we identified one (hereafter referred to as Vav-rtTA3) that showed particularly effective tet-regulated reporter expression in blood cells (see below).

In addition to four tet-on mouse strains, we tested two previously described transgenic strains that express the tet-off transactivator from the well characterised hematopoietic promoters Vav and Eμ. In contrast to the four tet-on strains, which were all maintained on a C57BL/6 background, the two tet-off strains were FVB/N strain background. Vav-tTA mice were originally developed to drive tet-regulated oncogene expression across the hematopoietic system [21]. Eμ-tTA mice express tTA under the control of the immunoglobulin heavy chain (IgM) enhancer and the SRα promoter [22]. The Eμ enhancer is particularly active in developing B and T lymphocytes [23], and effectively inducible vav cre lymphoid-specific expression in mouse models [24].

In all cases adult mice carrying transactivator and reporter transgenes developed normally and were analysed alongside littermate single transgenic or wild type controls. For tet-on strains, administering Dox food for one inducible vav cre prior inducible vav cre analysis activated reporter expression. GFP expression was determined in various hematopoietic cell types using flow cytometry of cells isolated from bone marrow, spleen, thymus and blood. We initially focused on stem cells and early progenitors of the lymphoid and myeloid lineages. Using well-defined surface antigens to identify cell types (Figure S2), we examined tet-regulated GFP reporter expression in populations of cells enriched for hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), megakaryocyte/erythroid progenitors (MEPs) and megakaryocyte progenitors (MkPs) (Figure 1). Notably, each transactivator strain showed a distinct GFP expression profile across these cell types. Of all the transactivator strains analysed, CAG-rtTA3 consistently drove highly efficient (near 100%) and intense GFP expression in HSCs and early progenitors, with the exception of MEPs (Figure 1). GFP expression inducible vav cre by CMV-rtTA was similar to CAG-rtTA3 but less uniformly intense, and approximately 10% of most progenitor cell populations in this strain failed to express GFP. The ROSA26-M2rtTA strain showed more heterogeneous GFP induction between and within different stem and progenitor populations, with poor expression intensity in HSCs compared with the CAG-rtTA3 and CMV-rtTA strains. However ROSA26-M2rtTA drove uniformly high reporter expression in MEPs, a cell type with weak or heterogeneous reporter expression in the other five strains examined (Figure 1). In keeping with the previously described pan-hematopoietic expression pattern of the Vav promoter [20], we observed reporter expression in all stem and progenitor cell types of Dox-treated Vav-rtTA3 bitransgenic mice and untreated Vav-tTA bitransgenic reporter mice (Figure 1). However both Vav promoter-driven strains showed a significant proportion of GFP– cells in each progenitor population, suggesting they are less efficient than CAG-rtTA3 and CMV-rtTA for inducible expression in these cell types. Intriguingly, Eμ-tTA transactivator mice drove appreciable GFP expression in HSCs and most progenitor populations despite predicted lymphoid-specific activity (Figure 1).

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Figure 1. Tet-regulated GFP vava baby monitor screen not working expression in hematopoietic stem cells and early progenitors of tet-transactivator transgenic mice.

Flow cytometry profiles of GFP expression in hematopoietic stem and progenitor cells isolated from the bone marrow of various transgenic mouse strains. Profiles from a representative mouse (n = 2 mice analysed per genotype) carrying the indicated transactivator transgene along with the TRE-GFP-shLuc reporter transgene are shown in green, with wild type controls shown in black. Tet-on inducible vav cre reporter mice (CAG-rtTA3, CMV-rtTA, ROSA26-M2rtTA, Vav-rtTA3) were given Dox food for 7 days before analysis, whereas tet-off bitransgenic reporter mice (Eµ-tTA and Vav-tTA) were untreated. The percentage of GFP+ cells in each population is indicated. Trane installation vav-in-19 Lin–Sca1+Kit+ (LSK) hematopoietic stem cell. CLP: Lin–Kit^IntSca1+CD127+ common lymphoid progenitor. CMP: Lin–Sca1–Kit+CD34+FcγRII/III– common myeloid progenitor. GMP: Lin–Sca1–Kit+CD34+FcγRII/III+ granulocyte/macrophage progenitor. MEP: Lin–Sca1–Kit+CD34–FcγRII/III– megakaryocyte/erythroid progenitor. MkP: Lin–Sca1–Kit+CD41+CD150+ megakaryocyte progenitor. Gating strategies are shown in Figure S2.

https://doi.org/10.1371/journal.pone.0054009.g001

Variable Transactivator Function in Differentiated Hematopoietic Cell Types

We also examined tet-regulated GFP expression patterns in more differentiated hematopoietic cell populations in the bone marrow, spleen, thymus, and blood. Once again CAG-rtTA3 was the most potent of the three ‘ubiquitous’ tet-on strains across more mature cell types, with 85–100% GFP expression in thymocytes, splenic B cells, bone marrow myeloid cells and blood platelets (Figure 2). Reporter expression in immature CD4+CD8+ (DP) thymocytes was efficient and intense in all strains apart from CMV-rtTA, suggesting that this cell type is particularly amenable to transactivator expression from different promoters. Strong reporter expression in Eμ-tTA DP thymocytes is consistent with a previous report where use of this strain to drive expression of TRE-Myc resulted predominantly in DP T cell lymphoma [22]. Notably, all strains showed a dramatic reduction in the efficiency of reporter expression in more mature, splenic CD8+ T lymphocytes (Figure 2; see below). Reporter expression in splenic B cells was also surprisingly poor in several strains, and only CAG-rtTA3 drove intense reporter expression in a high proportion of this cell population. We found that rtTA3 mRNA expression correlated with GFP expression in splenic B cells of Inducible vav cre TRE-GFP-shLuc mice (Figure S3), suggesting that transactivator expression level is an important determinant of reporter expression. Interestingly, GFP expression in splenic B cells of inducible vav cre Vav-tTA bitransgenic mice far outweighed that of their Dox-treated Vav-rtTA3 counterparts, potentially due to different Vav promoter activity based on transgene insertion site, transgene copy number, or strain background. CAG-rtTA3 drove intense and efficient GFP expression in bone marrow Gr1+Mac1+ myeloid cells (predominantly neutrophils), a cell population that showed very poor induction in most other strains (Figure inducible vav cre. The highly efficient reporter expression driven by CAG-rtTA3 in most cell types indicates that the Col1a1-targeted reporter transgene is amenable to transactivation across the hematopoietic system, and further implicates transactivator expression level as a critical determinant of reporter expression in different transactivator strains (see Discussion).

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Figure 2. Tet-regulated GFP reporter expression in developing and mature hematopoietic cells of tet-transactivator transgenic mice.

Flow cytometry profiles are as described in Figure 1. Thymocytes: CD4+CD8+ thymocytes. T cells: CD3+ splenocytes. B cells: B220+ splenocytes. Myeloid cells: Gr1+Mac1+ bone marrow cells. Platelets: CD41+ peripheral blood (plasma) cells. Profiles are from a representative mouse (n = 2–6 analysed per genotype).

https://doi.org/10.1371/journal.pone.0054009.g002

Inducible Reporter Expression in Megakaryocytes/platelets Correlates with Phenotype

Of all cell types examined, platelets showed the greatest variability in reporter expression between different transactivator strains. Platelets from CAG-rtTA3 and CMV-rtTA bitransgenic reporter mice uniformly expressed high GFP levels, whereas in other strains they were remarkably refractory to tet-regulated expression (Figure 2). Reporter expression in platelets was generally consistent with that in megakaryocyte precursors (Figure 1). To verify that GFP reporter expression accurately reflects transactivator function in platelets, we examined the phenotypic effects of tet-regulated knockdown of an endogenous gene in several transgenic transactivator strains. Bcl-x_L (Bcl2l1) is required for maintaining platelet survival in adult mice [25], and we have previously shown that tet-regulated Bcl-x_L knockdown in megakaryocytes causes severe thrombocytopenia [11]. We crossed TRE-GFP-shBcl-x_L transgenic mice [11] to several transactivator mice. Dox treatment of CAG-rtTA3; TRE-GFP-shBcl-x_L mice and CMV-rtTA; TRE-GFP-shBcl-x_L mice induced severe thrombocytopenia, with platelet levels falling to less than 10% of those in littermate controls or untreated mice (Figure 3). In contrast, Dox-treated ROSA26-M2rtTA; TRE-GFP-shBcl-x_L mice and untreated Vav-tTA; TRE-GFP-shBcl-x_L mice maintained normal platelet levels (Figure 3). These observations confirmed a close correlation between reporter expression and functional tet-regulated gene knockdown, and highlight marked differences in the ability of several ‘ubiquitous’ transactivator mouse strains to induce expression and associated phenotypes in a defined cell type.

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Figure 3. Transactivator-specific thrombocytopenia associated with Bcl-x_L knockdown in megakaryocytes/platelets.

Peripheral blood platelet counts of ROSA26-M2rtTA; TRE-GFP-shBcl-x_L, CMV-rtTA; TRE-GFP-shBcl-x_L, CAG-rtTA3; TRE-GFP-shBcl-x_L, and Vav-tTA; TRE-GFP-shBcl-x_L. Mice were either untreated (tet-off mice) or doxycycline treated for one week (tet-on mice) prior to blood sampling. Mice were bled between 6 and 14 weeks of age.

https://doi.org/10.1371/journal.pone.0054009.g003

Variable Tet-regulated Expression During T cell Differentiation in vivo

Having noted that reporter expression in several transactivator strains was far more efficient in developing, immature T cells in the thymus relative to mature T cell populations in the spleen (Figure 2), we tracked reporter expression during incremental stages of T cell development in these organs (Figure 4, Figures S4 and S5). The CAG-rtTA3 strain drove efficient and intense reporter expression throughout T cell development in the thymus, transitioning from CD4–CD8– progenitors through to more mature CD4+CD8– and CD4–CD8+ single positive (SP) populations (Figure 4A). In contrast, naïve SP T lymphocytes in the spleen of these mice only showed weak reporter expression (Figure 4B). This may be explained by poor CAG promoter activity and low rtTA3 expression in naïve T cells, however given that all transactivator strains displayed poor reporter expression inducible vav cre this cell type (Figure S4) we cannot rule out inaccessibility of the TRE-GFP-shLuc reporter transgene. Remarkably, high level GFP expression was restored in a large proportion of effector and memory T cells (Figure 4B). As most splenic T cells are inducible vav cre, the reporter expression observed in splenic T cell subsets is consistent with a preponderance of GFP-low cells in the total splenic T cell population vave health funding 2).

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Figure 4. GFP reporter expression in T cell subsets of CAG-rtTA3 mice.

Flow cytometry profile of GFP expression in thymic and splenic T cell subsets from vav shes mine representative CAG-rtTA3 bitransgenic reporter mouse (green) compared with a control mouse (black). Mice were given Dox food for 7 days before analysis. The percentage of GFP+ cells in each population is indicated. (A) Reporter expression during thymocyte differentiation through DN (CD4–CD8–) to DP (CD4+CD8+) to SP (CD4+CD8– and CD4–CD8+) stages. (B) Reporter expression in splenic T cell subsets. Naïve: CD62L+CD44–, Effector: CD62L–CD44+, Effector memory: CD44+CD127+CD62L–, Central memory: CD44+CD127+CD62L+. Gating strategies are shown in Figure S5.

https://doi.org/10.1371/journal.pone.0054009.g004

In vivo Kinetics of Vav bet chey tet vav tet and Tet-off Reporter Expression

A major strength of tet-regulated systems is rapid induction or repression of a protein-coding gene or shRNA. Having demonstrated particularly effective tet-regulated expression in DP thymocytes of Vav promoter-driven tet-on (Vav-rtTA3; TRE-GFP-shLuc) and tet-off (Vav-tTA; TRE-GFP-shLuc) mice (Figure 2), we investigated the in vivo kinetics of GFP induction and repression respectively in this cell population upon doxycycline treatment. Time course analysis revealed rapid reporter induction in Vav-rtTA3; TRE-GFP-shLuc mice, with over 30% of DP thymocytes expressing GFP after one day inducible vav cre Dox treatment (Figure 5A). Notably, after only 2 days of treatment approximately 60% of DP thymocytes were GFP+, most of which comprised a distinct GFP-high peak. The proportion of thymocytes expressing GFP reached near-maximum levels inducible vav cre after four days on Dox (Figure 5A). A ftuw vav high proportion of thymocytes inducible vav cre the corresponding untreated Vav-tTA; TRE-GFP-shLuc tet-off inducible vav cre mice expressed high GFP levels, which gradually diminished upon Dox treatment (Figure 5B). Approximately 70% of thymocytes remained GFP+ after 4 days of Dox treatment in vivo, albeit with low fluorescence intensity. In principle the half-life of a tet-regulated overexpressed protein (in this case GFP) dictates its rate of decay upon de-induction, and likely contributes to the slower rate of reporter repression relative to induction we observed in thymocytes (Figure 5A and 5B).

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Figure 5. Kinetics and variability of GFP reporter expression.

(A, B) Flow cytometry profiles of GFP expression in CD4+CD8+ thymocytes from representative Vav-rtTA3; TRE-GFP-shLuc (A) and Vav-tTA; TRE-GFP-shLuc (B) mice during a time course of Dox treatment. (C) Flow cytometry of GFP expression in peripheral blood B220+ B cells of four different Vav-tTA; TRE-GFP-shLuc mice. The percentage of GFP+ cells ranged from 92–97% as indicated.

https://doi.org/10.1371/journal.pone.0054009.g005

Discussion

Inducible gene over-expression or knockdown in mice using the tet-regulated expression system has proven very useful for inducible vav cre gene function in vivo. Although a multitude of tet transactivator transgenic mice have been developed, in many cases the pattern and level of transactivator expression and resulting TRE-driven protein or shRNA expression are poorly characterised. In this study we have used flow cytometry to systematically measure the tet-regulated expression of inducible vav cre fluorescent protein reporter across the hematopoietic system of several different tet transactivator mouse strains. This provided a unique opportunity to accurately quantitate not only the proportion of a particular cell type with reporter expression, but also expression level per cell based on fluorescence intensity.

A key finding of this study was the degree of heterogeneity in reporter expression in several mouse strains where transactivator expression is under the control of a nominally ubiquitous vava or vantrue such as CMV or ROSA26. This heterogeneity occurred at three levels. Firstly, reporter expression often varied markedly between different cell lineages within a transactivator mouse. For example CMV-rtTA reporter mice showed highly efficient GFP induction in the megakaryocyte/platelet lineage, however expression in myeloid and lymphoid cells was poor compared with other strains. Secondly, we observed differences in reporter expression between developmental stages within particular hematopoietic lineages. This was especially evident in the T cell lineage, where in all strains examined there was a dramatic fall in reporter expression in peripheral, naive T cell subsets compared with their immature progenitors in the thymus. In several strains we also noted decreased reporter expression in platelets compared with their megakaryocyte progenitors. These findings clearly caution against extrapolating reporter expression from one differentiation stage to another, and suggest that as progenitor cells proliferate and differentiate down a particular lineage the expression of tet-regulated transgenes can fluctuate significantly. This is an inducible vav cre drawback of regulated transcription systems, and contrasts with the ‘hit-and-run’ genetic changes induced using Cre/lox based mouse models. A third and particularly problematic feature of some transactivator mouse strains examined was significant variability in reporter expression between cells within an immunophenotypically defined cell population. In principle this variation can be explained by mosaic transactivator expression and/or accessibility of the TRE response transgene at the Col1a1 locus. This locus was originally chosen as a transgenic ‘landing pad’ because it supports transgene expression even in cell types that do not normally express Col1a1 [10]. However we note that Col1a1 is expressed at low but uniform levels across the wide range of mouse hematopoietic inducible vav cre types analysed in the “Immunological Genome Project” (www.immgen.org) [26]. Indeed our results revealed a remarkable, near-100% inducible vav cre of Inducible vav cre expression in most cell types of the CAG-rtTA3 strain, confirming that the Col1a1 locus is highly amenable to reporter transactivation across the hematopoietic system. This is consistent with our previous observations in other tissues [27]. An exception is naïve, splenic T cells, in which we noted poor reporter expression in all transactivator strains. This raises the possibility of silencing of the Col1a1-targeted tet responsive transgene in this cell type, perhaps associated with the high degree of chromatin condensation in naïve T cells relative to activated/memory T cells [28]. Previous retroviral and transgenic studies have also noted low level expression of CMV-based promoters in the T cell lineage [29]–[31].

Our results suggest that sub-optimal transactivator protein expression limits reporter induction in the ‘ubiquitous’ transactivator strains CMV-rtTA and ROSA26-M2rtTA. The activity of transgenic promoters lacking normal regulatory elements can be subject to position effects associated with epigenetic silencing [32]. Va-lt002 vava ROSA26-M2rtTA is a knockin transgene that makes use of the endogenous ROSA26 promoter, therefore the basis of variable transactivator expression within certain cell types of this strain remains unclear.

Of the six transgenic strains tested, CAG-rtTA3 was the most effective driver of reporter expression across inducible vav cre hematopoietic cell types. This was exemplified by high efficiency inducible vav cre expression in bone marrow myeloid cells and platelets, cell types refractory to tet-regulated expression in most if not all other strains. Efficient expression across almost all hematopoietic cell types is consistent with our previous observations in non-hematopoietic tissues [9], and establishes the CAG-rtTA3 strain as a highly effective driver of tet-regulated expression across the great majority of cell types examined. However rani ki vav gujarat india map location noted poor CAG-rtTA3-driven reporter expression in MEPs and a significant proportion of splenic B cells, emphasising the need for validation of transactivator function in cell types of interest even within broadly effective transactivator strains.

It is often desirable to restrict tet-regulated expression to the hematopoietic vavá cantor. For example, previous models of oncogene addiction or tumour suppressor hypersensitivity in leukemia and lymphoma have relied on transgenic mice where transactivator expression is controlled by the largely hematopoietic-specific Eµ or Vav promoters [8], [21], [22]. Although our results suggest that Vav-tTA, Vav-rtTA3, and Eµ-tTA transgenic mice display robust tet-regulated reporter expression in many hematopoietic cell types, expression in stem, progenitor, and myeloid cells of these strains was clearly sub-optimal relative to the highly effective CAG-rtTA3 strain. In principle, efficient yet hematopoietic-specific tet-regulation can be achieved by transplanting bone marrow or fetal liver-derived hematopoietic stem cells derived from CAG-rtTA3 or other effective ‘ubiquitous’ promoter-driven transactivator mice into lethally irradiated wild type mice. The resulting chimeric reconstituted mice should allow robust Dox-dependent expression across the hematopoietic system with minimal effects on non-hematopoietic tissues.

While highlighting the potential heterogeneity of tet-regulated expression across the hematopoietic system in vivo, our study also emphasises the power of fluorescent reporter flow cytometry for analysis and isolation of cells with optimal tet-regulated inducible expression from a mixed inducible vav cre. For tet-regulated overexpression of a gene of interest, reporter co-expression can be achieved using bicistronic expression cassettes based on internal ribosome entry sites (IRES) or 2A peptides [33]. Similarly, tet-regulated gene knockdown can be tracked by including shRNA sequences in the 3′ UTR of the reporter transcript. The TRE-GFP-shRNA configuration used in this study has been optimised for ty vava GFP expression and target gene knockdown [9].

In summary, we have systematically characterised tet-regulated expression across the hematopoietic system of several transactivator mouse strains. The reporter expression patterns described here allow inducible vav cre informed choice of the most appropriate transactivator transgenic strain for investigation of a certain hematopoietic cell type or process, and provide a basis for the accurate interpretation of hematopoietic phenotypes generated using these strains. Furthermore we have found that CAG-rtTA3 transgenic mice facilitate intense and efficient inducible gene regulation across the majority of blood cell types, establishing this strain as a valuable tool for the study of hematopoietic development and disease.

Methods

Supporting Information

Acknowledgments

We thank S. Best, R. Lane, L. Tuohey, and J. Corbin for technical assistance; T. Willson for generating the Vav-rtTA3 construct; M. Salzone, M. Dayton, P. Kennedy, K. Stoev, and Inducible vav cre Bioservices staff for mouse inducible vav cre D. Hilton, W. Alexander, and E. Major for ES cell and mouse resources; J. Adams for the HS21/45 Vav transgenic vector; and E. Viney and J. Sarkis at the Australian Phenomics Network Transgenic RNAi service. We thank H. Varmus and F. Cong for CMV-rtTA mice, R. Jaenisch for ROSA26-M2rtTA mice, D. Largaespada for Vav-tTA mice, and D. Felsher for Eμ-tTA mice. We thank members of the Dickins laboratory for advice and discussions.

Author Contributions

Conceived and designed the experiments: MT BTK SJT SWL DCSH RAD. Performed the experiments: MT LED JEP CLC. Analyzed the data: MT JEP CEC BTK SJT DCSH RAD. Contributed reagents/materials/analysis tools: LED SWL. Wrote the paper: MT RAD.

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• Francesca Grespi, 
• Eleonora Ottina, 
• Nikolaos Inducible vav cre
• Stephan Geley, 
• Andreas Villunger

x

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 inducible vav cre, 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 inducible vav cre 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.

1) Insertion of lacO elements into the VavP vector confers IPTG sensitivity

We chose to adopt the Vav-gene promoter, demonstrated to confer tissue specific expression of transgenes in vivo [6], in order to make it amenable to timed regulation. A Vav plastics yelp element derived from the pOPI3CAT expression vector containing three symmetric lac operator (lacO) binding sites recognized by the lac repressor (lacI), 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 VavP-transgenic vector. Venus, an optimized version of GFP [12], was inserted as a reporter into the MCS (Fig. 1a). The insertion was confirmed by sequencing and the VavlacOVenus (VLV) construct was tested for functionality by transient transfection into 293T cells, either alone or together with a plasmid encoding the codon optimized mammalian version of the lacI[10]. Inspection of cells in an immunofluorescence microscope followed by flow cytometric analysis confirmed expression of Venus in a subset of cells transfected with the VLV plasmid alone that was reduced when co-expressed along with a lacI-encoding plasmid (Figs. 1b,c). Quantification by flow cytometry revealed that co-expression of lacI indeed reduced the percentage of Venus⁺ cells from ∼24% to ∼10%, while addition of the synthetic inducer IPTG lead to a significant (p<0.05) increase in Venus expression (Fig. 1c). Increasing the concentration of IPTG further to 0.1 or 1 mM did not significantly improve the efficacy of re-induction, consistent with published results using embryonic R3 cells [10]. Venus encoding cDNA was also subcloned into an unmodified version of the Vav-transgenic vector (VV) that served as a control. The percentage of Venus expressing 293T cells was comparable between VLV and VV transfected cells (Fig. 1c) demonstrating inducible vav cre the insertion of the SV40-intron did not compromise the activity of the Vav promoter, at least in vitro.

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Figure 1. Generation of an IPTG-responsive version of the Vav-transgenic vector.

(a) Schematic representation of the modified VavP HS21/45 plasmid (VLV) where three lac repressor binding sites (lacO, sequence in red) were introduced in the SV40 intronic sequence upstream of the multiple cloning site, bearing the Venus cDNA. (b) Functionality of the construct was assessed before oocyte-injection by transient transfection into 293 T cells in the presence or absence of a construct encoding for the lacI repressor followed by flow cytometric analysis. (c) 293 T cells transfected with empty vector, VV, VLV ± lacI were exposed to different doses of isopropyl β-D-1-thiogalactopyranoside (IPTG) and vava voom 24 reviwrs h later the percentage of Venus⁺ cells was quantified by flow cytometric analysis. Bars represent means ± SEM of a representative experiment performed in triplicate *p<0,01, #p<0.05.

https://doi.org/10.1371/journal.pone.0018051.g001

2) Generation of VavLacOVenus transgenic mice

Transgenic mice were generated by microinjection of the VLV or VV constructs isolated as a linear fragment into fertilized oocytes from FVB mice. PCR genotyping on tail DNA using primers specific for Venus cDNA and VavP cassette sequence was performed to identify founders carrying the transgene (Fig. 2a). Since transgene insertion does not necessarily result in its expression, e.g. due to silencing or positioning effects in heterochromatin, we collected peripheral blood (PB) from PCR⁺ and PCR⁻ mice and performed flow cytometric analysis to verify the typing results and quantify the percentage of Venus-expressing cells. Venus expression in the peripheral blood varied significantly between founders, indicating that some of the founders were mosaic or may show variegated transgene expression e.g., vava or vantrue to positioning effects of the transgene (Fig. 2b,c). Out of the transgene-expressing founders, we chose those showing highest Venus expression for further breeding, i.e. VLV A1 and A3 as well as Inducible vav cre A9 and A19. All experiments shown below are derived using VLV A3 and VV A9 derived progeny, respectively. Similar results regarding heterogeneous transgene expression were also observed in the offspring of other founders (not shown).

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Figure 2. Identification of transgenic mice.

The VavLacOVenus (VLV) plasmid as well as an unmodified version of the VavP plasmid encoding Venus cDNA (VV) were digested with AseI and PvuI and an approximately 9.2 kb AseI-PvuI fragment was used for microinjection into fertilized FVB oocytes. (a) Transgenic founders were first identified by PCR genotyping on tail DNA. VLV  =  VavLacOVenus transgenic mouse, VV  =  VavVenus transgenic mouse, WT  =  wild type FVB mouse. (b) Histograms from flow cytometric analysis free download vavavo vave vannummakal sammanam different levels of transgene expression in PCR-typed founders. (c) Range of transgene expression levels in PCR⁺ founders, quantified as in (b).

https://doi.org/10.1371/journal.pone.0018051.g002

3) Expression of Venus protein is detectable in myeloid and lymphoid cells but appears variegated

Although the transgenic animals did not show any overt phenotype up to an observation period of 6 month, we wanted to monitor whether overexpression of Venus could have some impact on lymphocyte number or survival, since high level expression of GFP has been reported to cause some toxicity in cultured cells [13] and reportedly correlated with premature lethality when overexpressed strongly in cardiomyocytes [14]. First, we performed Western blot analysis on different tissues that confirmed restriction of transgene expression to haematopoietic organs (Fig. 3a). Next, inducible vav cre quantified leukocyte numbers in haematopoietic organs and compared transgenic lines with littermate controls that failed to reveal any significant differences (p>0.2) in cell number (Fig. 3b). Second, we put primary lymphocytes derived from thymus, spleen or lymph nodes in culture and monitored cell survival by Annexin V/PI staining, in combination with cell surface marker staining to identify T and B cells, over time. Thymocytes as well as mature B- and T-cells derived from the spleen of the VLV or VV mice did not show any difference in survival in culture when compared to those ones derived from wt mice (Fig. 3b). Unexpectedly, the B-cells derived from the lymph nodes of VLV mice appeared more resistant to spontaneous apoptosis than the ones of VV or wt mice that died with similar kinetics (Fig. 3b). Together our results show that Venus expression is well tolerated in lymphocytes over time in vivo. Also, in the VLV strain chosen for detailed analysis, transgene insertion may influence expression/function of gene(s) associated with the survival of mature B cell, at least in vitro. Inducible vav cre, since we did not observe B cell accumulation in vivo this observation was not followed up in detail.

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Figure 3. Characterization of Venus expression, organ cellularity and lymphocyte survival in vitro.

(a) Protein lysates (55 µg/lane) from the indicated organs harvested from wt or VLV mice were separated by SDS-PAGE, transferred onto nitrocellulose membranes and immunoblotting was performed using an antiserum generated against GFP. Membranes were reprobed using a monoclonal antibody recognizing GAPDH to demonstrate comparable protein loading. *MLN  =  mesenteric lymph nodes. (b) Mice of the indicated genotpyes 6-8 weeks of age were sacrificed inducible vav cre evaluate organ cellularity (n = 4/genotype). Primary cells derived from total spleen, thymus or lymph nodes were put in culture and spontaneous apoptosis was quantified by flow-cytometry. Markers for T- (CD3) and B-cells (CD19) were used in combination with Inducible vav cre staining to monitor survival over time. Data points represent means ± SEM of three independent experiments (§  =  p>0.001 VLV vs VV and WT, #  =  p>0.01 VLV vs VV and WT).

https://doi.org/10.1371/journal.pone.0018051.g003

We continued our analysis quantifying the percentage of Venus⁺ cells in different primary and secondary lymphatic organs. Therefore, we stained single cell suspensions with antibodies specific for different cell surface markers, identifying T-cells, B-cells or myelocytes and performed flow cytometric analysis. Venus⁺ cells were found in all the leukocyte subpopulations tested. However, the relative percentage of Venus⁺ cells varied between the individual transgenic lines as well as between littermates, indicating variegated expression of the reporter or mosaicism due to stochastic gene silencing. Similar observations were made in all other lymphoid organs analyzed (Fig. 4a). In VLV transgenic mice, T cells showed Venus expression in all the organs ranging from 35%–80%, with highest expression found in CD8⁺ T cells in lymph nodes and spleen (∼80%), while the percentage of Venus⁺ CD4⁺ T cells was frequently lower in thymus, peripheral blood, spleen, lymph node and bone marrow (p<0.03) (Fig. 4b). In the CD19⁺ B cell compartment in the periphery, we found that transgene expression was actually highly comparable between spleen, peripheral blood and bone marrow with 70–80% of Venus expressing B cells, but only inducible vav cre half of the B cells in the lymph node were expressing the transgene (Fig. 4b). Immature pro- and pre-B-cells in the bone marrow also expressed Venus, with a slightly higher percentage of transgene positive pro-B than pre-B cells (Fig. 4b). The percentage of Venus⁺ Mac1⁺ myelocytes was comparable to the percentage of Venus⁺ lymphocytes in the spleen, while it was significantly lower in the bone marrow and peripheral blood (p<0.002) of VLV mice (Fig. 4b). Comparing levels of Venus⁺ cells in the VLV with those in VV transgenic mice we noticed an overall similar pattern of transgene expression but inducible vav cre generally lower percentage of Venus⁺ cells in the VV strain (Fig. 4b). This phenomenon is most likely due to different sites of inducible vav cre of the transgene and/or copy number variation. Notably, qPCR analysis performed on tail DNA derived from three randomly picked animals of each strain revealed an about 2.5-fold higher signal for Venus in the VV samples, indicating higher copy number in this strain (not shown). This suggests that chromatin effects at the site of integration rather than copy number accounts for the difference in transgene expression between the two strains.

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Figure 4. Characterization of transgene expression in haematopoietic cells from VLV and VV mice.

Animals that proved positive for Venus expression in the peripheral blood were sacrificed for further analysis of transgene expression in different organs. (a) Single cell suspension from spleens were stained with fluorochrome labeled antibodies specific for cell surface markers identifying T cells (CD4, CD8), B cells (CD19), as well as myelocytes (Mac-1) and subjected to flow cytometric analysis. Representative dot blots showing variegate expression of the transgene are shown. (b) Summary of the experiments shown in (a), performed to assess the percentage of transgene expressing leukocytes in all major haematopoietic organs. Bars represent means ± SEM (wt n = 8, VLV n = 5, VV = 3); *p<0.001; #p<0.05. P values refer to significant differences in Venus expression between cells derived from VLV and VV mice. F2 generation offspring of VLV founder A3 and F3 generation offspring of VV founder A9 were used for this analysis, as well as transgene-negative littermate controls of both strains.

inducible vav cre https://doi.org/10.1371/journal.pone.0018051.g004

4) Gene-silencing of Venus expression in VavLacOVenus/LacI double transgenic mice

After having characterized Venus expression in the single transgenic mice, we started to cross VLV mice with mice transgenic for lacI. In these animals the Lac repressor protein is ubiquitously expressed from the human β-actin promoter, with high levels of repressor protein detected in the spleen [10]. First we started to analyze if Venus expression was shut down effectively in the peripheral blood of double-transgenic inducible vav cre identified by PCR genotyping, using flow cytometric analysis and whether it was re-inducible in culture. Venus expression in the peripheral blood dropped to ∼5% in double-transgenic animals, indicating effective shut down of transgene expression. We were also able to re-induce Venus expression vava or vantrue IPTG treatment in a significant portion of the cells (p<0.05), monitored up to 72 h and reaching plateau after 48 h with up to 30% of the cells re-expressing Venus (Fig. 5a). The dose-response behaviour of the cells suggested that the system was already maximally induced with 10 µM of IPTG, but Venus expression levels appeared more stable at later time-points when ≥50 µM IPTG were used, the concentration we chose for further experiments. When monitoring the inducible vav cre of mitogen-induced Venus⁺ T or B cells blasts derived from peripheral blood over time by gating on TCRβ⁺ T or CD19⁺ B cells, respectively, no significant difference was observed between the cell types (p = 0.23), but we were unable to reach the percentage of Venus⁺ cells observed in the peripheral blood from single transgenic VLV mice (Figs. 5b–d). Extended culture of cells was not increasing the percentage of Venus⁺ cells, probably due to the high rate of cell death occurring in the T and B cell blasts over extended times ex vivo which ultimately associated inducible vav cre loss of Venus expression (not shown).

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Figure 5. Transgene silencing and re-expression of transgene expression in vitro.

Primary cells derived from peripheral blood of VLV/LacI double transgenic mice were put in culture and stimulated with graded doses of IPTG. FACS analysis was performed at vava or vantrue indicated time points to monitor levels of Venus⁺ cells (a). Peripheral blood lymphocytes from mice of the indicated genotypes were stimulated with IL-2+PMA+ionomycin to drive T-cell activation or IL-2+IL-4+IL-5+LPS to drive B-cell proliferation. Proliferating cells were treated with 50 µM IPTG inducible vav cre day 3 after mitogen stimulation and levels of Venus⁺ cells were inducible vav cre by FACS analysis on all cells (b), as well as T-cells (c) or B-cells (d). Results from four independent experiments and 4-9 animals/genotype (wt = 5, Inducible vav cre n = 5, VLV n = 4, VLV/LacI n = 9) are represented as box plots. Box length equals interquartile range. Circles represent minimal and maximal values; *p<0.001; #p<0.05. F2 generation offspring of VLV founder A3 were crossed with LacI mice. Single- double- and non-transgenic littermates were used for analysis.

vava 76ers, we aimed to verify whether the system was inducible in inducible vav cre. First, we started to treat the animals with 10 mM IPTG in the drinking water, as suggested by previous results using a regulated version of the tyrosinase gene [10]. However, after monitoring Venus expression after 1 and 3 weeks of IPTG-treatment, respectively, we were unable to detect a significant portion of treatment-induced Venus⁺ cells in the peripheral blood of these animals. In line with our organ analysis, CD8⁺ T cells that showed highest levels of transgene expression (Fig. 4), showed best induction in a subset of cells (up to 10%), while induction in other T and B cell subset was detected in less than 5% of the cells (data not shown). So we decided to treat the mice with a single dose of IPTG i.p. In a pilot experiment, we treated 8 weeks old animals with graded doses of IPTG (0.1, 1 or 10 mg) and monitored the percentage of Venus⁺ cells in peripheral blood over time. As in our in vitro experiments, no substantial differences were noticed between the three doses tested and 1 mg IPTG was chosen for further experiments (data not shown). We collected peripheral blood from the tail vein of treated animals in different intervals and stained cells with antibodies specific for different cell surface markers identifying myelocytes, T and B cells. Similar to our in vitro experiments we were able to detect a significant increase (p<0.05) in percentage of Inducible vav cre cells in the peripheral blood of double-transgenic mice, with CD8⁺ T cells and IgM⁺D⁻ naïve B rani ki vav gujarat india map location showing the best response while Mac-1⁺ myelocytes only responded poorly, if at all (Fig. 6). However, variation in gene silencing was high with CD8⁺ T cells showing the highest leakiness in double transgenic mice. In none of the cell types analyzed were we able to induce Venus expressing to the levels noted in single transgenic mice (Fig. 6).

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Figure 6. Transgene re-expression in vivo.

Mice of the indicated genotypes were treated i.p. with 1 mg IPTG and levels of Venus⁺ cells in T- B- and myeloid subsets in the peripheral blood were quantified by FACS analysis following the indicated times. Results of three independent experiments and 3 (VLV and LacI) or 6 (VLV/LacI) animals/genotype are represented as box plots. Box length equals interquartile range. Circles represent minimal and maximal values; *p<0.001; #p<0.05. F2 generation offspring of VLV founder A3 were crossed with LacI mice. Single- double- and non-transgenic littermates were used for analysis.

https://doi.org/10.1371/journal.pone.0018051.g006

Discussion

We adopted the expression plasmid VavP21/45, containing the promoter of the Vav gene, to drive expression of a fluorescent reporter protein, Venus, under the control of the lac operator in vivo. We confirmed functionality and regulation of reporter expression after insertion of lacO binding sites by LacI after IPTG treatment lettewr vav pictures 293T human embryonic kidney cells (Fig. 1). In the presence of a co-transfected Lac repressor, the percentage of Venus⁺ cells was reduced but not completely shut down, consistent with previous results ectopically expressing a lacO-modified version of the human huntingtin promoter driving firefly luciferase [10], [15]. Initially, we ascribed the limited expression and silencing to the fact that a heterologous cell system was used where the Vav-gene is usually not active [6]. Also, the addition of IPTG inducible vav cre the cell culture media only partially restored original levels of Venus expression in 293T cells, which has been noted before when using this system in mammalian cells [16]. Incomplete shut-down of transgene expression might be related or due to the spacing and positioning of the lacO elements in close proximity and short distance within the pOPI3-CAT-derived SV40 intron, upstream of the multiple cloning site. It has been reported before, that two lacO sites positioned on either site of the transcription start site are sufficient to confer regulation to mammalian promoters by lacI and IPTG, while the third binding side is usually positioned further up or downstream and mainly serves to increase the local concentration of lacI [11]. However, since we did not want to interfere with the tissue-specificity of the VavP promoter-construct, we were prepared to accept reduced promoter regulation in our initial in vivo approach using Venus as a reporter.

When assessing transgene expression in peripheral blood of the founders, we found a high variation rani ki vav gujarat india map location the percentage of Venus⁺ cells (Fig. 2), a possible consequence of the microinjection technique used to generate transgenic animals, where copy number and insertion sites can vary and mosaicism is frequently observed due to transgene insertion at the two cell stage or later. However, consistent with the heritable nature of position effect variegation (PEV) [17], transgene expression was also observed to vary in F2 progeny of selected founders. Based on our organ analysis, this inducible vav cre to vary strongly between cell types and tissues (Fig. 4). Our results resemble those reported initially when using the bacterial β-gal gene as a reporter in the context of the VavP promoter that was expressed in a variegated manner and subjected to frequent silencing over time [18]. PEV is a well known phenomenon in transgenesis depending on chromatin structures near the site of insertion, most pronounced when transgene constructs are used that lack a defined locus control region inducible vav cre, but inducible vav cre hard to distinguish in our case from inducible vav cre silencing-induced mosaicism. Based on experiences using hCD4 or hBcl-2 as transgenes in the VavP vector showing uniform expression of the human transgenes tested [6], [7], we can only speculate that the non-mammalian Venus sequence, although codon optimized for the use in mammalian cells, may be susceptible to stochastic epigenetic silencing in a certain percentage of cells, as was observed previously when VavP-lacZ tg mice were compared to VavP-hCD4 tg mice [6], [7]. Alternatively, the pOPI3-CAT derived SV40-derived intronic sequence containing the lacO sites may be subjected to epigenetic silencing, as noted before when bacterial or viral sequences were introduced into mammalian genome [19]. However, the latter seems unlikely, since we noted similar patterns of transgene expression in the VV strain, lacking this DNA element (Fig. 5). Since we were forced to use different restriction enzymes to linearize the modified VLV plasmid, it remains also possible that the residual bacterial DNA sequence of about 250 base pairs that we could no longer remove (Fig. 1a) may render the transgene more susceptible to silencing. Alternatively, high-copy number insertion of transgenes reportedly favours variegation of expression due to repeat-induced gene silencing [17] which may in part explain why VV mice, that do have a higher copy number insertion than VLV mice based on qPCR analysis (not shown), showed stronger variegation (Fig. 4). Alternatively, high-level expression of Venus may not be well tolerated in haematopoietic cells vava eyewear price silencing of transgene expression over time or counter selection in stem cells. High-level GFP expression was reported to induce apoptosis in cultured cells and cardiomyopathy was observed in FVB mice with high-level expression of GFP when driven from the cardiac alpha-myosin heavy chain promoter [14]. However, our in vitro analysis did not show increased cell death in Inducible vav cre expressing lymphocytes (Fig. 2) and counter selection due to toxicity in leukocytes can be largely excluded since we did not see a significant reduction of the percentage of Venus⁺ cells in peripheral blood over time (not shown).

When we crossed our single transgenic VLV mice with the LacI repressor mice we noticed inducible vav cre reporter transgene expression was actually quite effectively silenced in the peripheral blood where only about 5% of cells scored as Venus⁺ by highly sensitive flow cytometry. In addition, emission of Venus fluorescence is significantly stronger as the one emitted by GFP [12], which initially suggested that silencing is very effective and well in the range or even better as when the Bujard system was used in vivo[20]. This background expression of Venus did not differ inducible vav cre between cell types of the peripheral blood (Fig. inducible vav cre making broad differences in lacI expression across the different haematopoietic cell types analyzed unlikely. However, when testing for the ability to re-induce transgene expression in vitro, we noted that we were unable to reach more than inducible vav cre Venus⁺ T or B cell blasts (Fig. 5). While this may have been due to high rates of cell death that correlates with loss of Venus expression in primary inducible vav cre upon extended in vitro culture, application of IPTG in the drinking water also failed to drive transgene re-expression in peripheral blood (not shown). This may be due to vava or vantrue reported short half-life and rapid clearance of IPTG from the peripheral blood stream by excretion via filtration in the kidneys [21] but should not be an issue in vitro. Similarly, when injecting mice with IPTG i.p., we also noted a very limited response, combined with considerable leakiness in different cell types that again differed between individual animals (Fig. 6). Notably, either repression was good, i.e. less than 10% of the cells were Venus⁺ then re-induction was also poor (20–30%), or the system was leaky (>20% Venus⁺ cells) allowing high levels of re-induction (up to 80%, e.g. in CD8⁺ T-cells). This opens the possibility that vav ayno lost voice inefficient shut down and leakiness observed may be related or due to inefficient expression of lacI by the β-actin inducible vav cre in different haematopoietic cell types. In previous studies characterizing LacI tg mice the levels of LacI protein expression was only assessed in tissue lysates but not at the cellular level [10], [15], leaving the possibility that its expression may actually be also not uniform across cell types within tissues. Alternatively, the Vav-gene promoter that shows differences in activity in between leukocyte subsets and is highly susceptible to epigenetic regulation [18] may either be too weak to overcome the repressive effect of LacI in all cells of a given lymphocyte subset before clearance of the inducer, or, become more easily subjected to heterochromatinization e.g., due to lacI-mediated inefficient transgene expression, inducible vav cre such events in some type of negative feed back loop. However, this needs to be formally demonstrated.

Our findings using the lacO/lacI system in haematopoietic cells differ from the successful use of this inducible vav cre driving tyrosinase expression in pigment-producing melanocytes, suggesting that these cells are able to accumulate more inducer and maybe retain higher level/constant expression of LacI. Furthermore, re-expression of the transgene in a subset of hair follicle stem cells or derived melanocytes may actually be sufficient to restore pigmentation [10].

Although, the lacO/lacI system in the current version tested shows only limited suitability for regulated transgenesis in vava or vantrue cells, after optimization, e.g., of lacO element number/positioning and/or reduction of transgene copy number, it may still be useful for certain applications. Variegated or mosaic expression of Venus may even be exploited to target expression of pro-apoptotic genes, recombinases or transforming oncogenes only to a subset of cells, e.g., by inserting inducible vav cre IRES-element in the 3′UTR of Venus, followed by the cDNA of interest. Alternatively, the same trick can be exploited to introduce an RNA interference based gene-silencing cassette, eliminating expression of a target gene in who built rani ki vav subset of cells.

Materials and Methods

Acknowledgments

We want to thank all our lab-members for insightful discussion, Irene Gaggl for mouse genotyping, Claudia Soratroi for technical assistance and Kathrin Rossi for animal care. We thank Prof. J. Adams for the VavP transgenic vector and Prof. H. Scrable for lacI transgenic mice.

Author Contributions

Analyzed the data: FG EO NY SG AV. Contributed reagents/materials/analysis tools: SG. Wrote the paper: FG Inducible vav cre. Designed research: SG AV. Performed research: FG EO NY.

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