Generation and evaluation of an IPTG-regulated version of Vav-gene promoter for mouse transgenesis

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.

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 24 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.

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 showing different levels of transgene expression in PCR-typed founders. (c) Range of transgene expression levels in PCR⁺ founders, quantified as in (b).

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 to 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 AnnexinV-APC+7-AAD 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).

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.

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 the 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 on day 3 after mitogen stimulation and levels of Venus⁺ cells were quantified 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, LacI 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.

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.