Strict control of transgene expression in a mouse model for sensitive biological applications based on RMCE compatible ES cells

Abstract

Recombinant mouse strains that harbor tightly controlled transgene expression proved to be indispensible tools to elucidate gene function. Different strategies have been employed to achieve controlled induction of the transgene. However, many models are accompanied by a considerable level of basal expression in the non-induced state. Thereby, applications that request tight control of transgene expression, such as the expression of toxic genes and the investigation of immune response to neo antigens are excluded. We developed a new Cre/loxP-based strategy to achieve strict control of transgene expression. This strategy was combined with RMCE (recombinase mediated cassette exchange) that facilitates the targeting of genes into a tagged site in ES cells. The tightness of regulation was confirmed using luciferase as a reporter. The transgene was induced upon breeding these mice to effector animals harboring either the ubiquitous (ROSA26) or liver-specific (Albumin) expression of CreER(T2), and subsequent feeding with Tamoxifen. Making use of RMCE, luciferase was replaced by Ovalbumin antigen. Mice generated from these ES cells were mated with mice expressing liver-specific CreER(T2). The transgenic mice were examined for the establishment of an immune response. They were fully competent to establish an immune response upon hepatocyte specific OVA antigen expression as indicated by a massive liver damage upon Tamoxifen treatment and did not show OVA tolerance. Together, this proves that this strategy supports strict control of transgenes that is even compatible with highly sensitive biological readouts.

Figure 1.

Strategy to create a platform ES cell line for RMCE-based ROSA26 targeting. (A) Strategy to make ubiquitously expressed ROSA26 locus RMCE accessible. Structure of the wild-type ROSA26 locus, the tagging vector harboring the heterospecific FRT sites and the targeted RMCE compatible locus after homologous recombination (HR) is depicted in the above figure. SA, splice acceptor site; F, wild-type FRT site; F5, mutant F5 site; Δneo pA; start-codon deficient neomycin phosphotransferase gene with polyadenylation signal; Rosa5′/3′, ROSA26 genomic flanking sequences; PAC, puromycin N-acetyltransferase gene; LUC, luciferase; L, wild-type loxP sites (inversely oriented); HR, homologous recombination; X, XbaI restriction site; DTA, Diphtheria toxin A gene. Shaded boxes indicate the exons. (B) Targeted integration of expression cassettes of choice into RMCE compatible ES cells via Flp-mediated cassette exchange. The above figure depicts the ‘tag and target’ strategy to integrate different expression cassettes of choice in the ROSA26 chromosomal background. In the RMCE permissible ROSA26 locus, the two non-interacting FRT sites flank the entire expression cassette followed by a 5′-truncated, ATG start codon defective neomycin phosphotransferase gene. The tagged parental ES cells are G418 sensitive. Co-transfection with the Flp recombinase expression plasmid and the targeting vector harboring the corresponding identical heterotypic FRT sites will lead to site-directed recombination via F and F5 as indicated by the crosses. After recombination, the defective Δneo gene is complemented by the IRES element and the ATG start codon positioned in-frame thereby rendering the cells undergoing the correct exchange event G418 resistant. The gene of interest (for example the ovalbumin antigen) is also inversely oriented flanked by oppositely oriented loxP sites. GOI, gene of interest; Flp, Flp recombinase; RMCE, recombinase mediated cassette exchange; L, wild-type loxP site (inversely oriented); IRES, encephalomyocarditis IRES. (C) Activation of the floxed GOI/LUC in presence of Cre. Here the gene of interest (GOI) was placed in the reverse orientation with respect to ROSA26 transcription and flanked by loxP sites oppositely oriented to each other. Hence this makes the GOI Cre activatable.

Figure 2.

Targeted integration of different antigen/gene cassettes into the parental FRT tagged ROSALUC mES cells via RMCE. The above figure gives a summary of the efficiency of integrating different targeting constructs into the tagged ROSA26 locus by Flp-mediated cassette exchange. Different expression cassettes were cloned into the pEMTAR backbone vector (28) harboring the heterotypic FRT sites along with the IRES element and the ATG start codon. These targeting vectors were used for subsequent cassette exchange in the RMCE compatible ROSA26 locus. Correct targeting was proven by PCR and/or Southern blot. LUC, luciferase; rTA, reverse tetracycline dependent transactivator; Tet, tetracycline dependent promoter; eGFP, enhanced green fluorescent protein; HBsAg, Hepatitis B surface antigen; OVA, ovalbumin; TAg, SV40 large T antigen; CAGGS, chicken β-actin promoter with cytomegalovirus enhancer; LTR, long terminal repeat; TAK, TAK protein; F, wild-type FRT site; F5, mutant F5 site; filled arrow head, wild-type loxP site; open arrow head, mutant loxL3 site.

Figure 3.

In vivo activation of Cre-dependent luciferase expression in bitransgenic ROSALUC X ROSA26-CreER^T2 mice. (A) In vivo non-invasive bioluminescent imaging (BLI) of ROSALUC X ROSA26-CreER^T2 offsprings. (a) BLI image of non-induced animals. Four-weeks-old bitransgenic ROSALUC X ROSA26-CreER^T2 mice along with single transgenic ROSA26CreER^T2 and ROSALUC as controls are indicated. (b) BLI image of animals after Tam induction. Image was acquired 5 days after the last Tam feed. Color bar indicates photons/cm²/s/steradian with the minimum and maximum threshold values. (B) Monitoring luciferase expression in the different organs isolated from double transgenic ROSALUC X ROSA26-CreERT² mice. The 4–8-weeks-old bitransgenic ROSALUC X ROSA26-CreERT² mice (induced and uninduced) were sacrificed and various organs were isolated. Tissue lysates obtained were subjected to a luciferase assay. The luciferase activity observed in RLU was normalized to micrograms of total protein present in the tissue sample. Figure depicts induced ROSALUC X ROSA26-CreERT². Non-induced ROSALUC X ROSA26-CreERT² as well as the control single transgenic ROSALUC and ROSA26-CreERT² mice showed an average of <2 RLU/μg of total protein and are not depicted in the figure. For each group four mice were analyzed.

Figure 4.

Monitoring luciferase expression in the different organs isolated from ROSAConL mice. The 4–8-weeks-old ROSAConL mice were sacrificed and various organs were isolated. Tissue lysates obtained were subjected to a luciferase assay. The luciferase activity observed in RLU was normalized to micrograms of total protein present in the tissue sample. Number of mice analyzed = 6.

Figure 5.

In vivo activation of Cre-dependent luciferase expression in bitransgenic ROSALUC X Alb-CreER^T2mice. (A) In vivo non-invasive bioluminescent imaging (BLI) of ROSALUC X Alb-CreER^T2 mice offsprings. (a) BLI image of animals not induced with Tam. Four-weeks-old bitransgenic ROSALUC X Alb-CreER^T2 mice along with single transgenic Alb-CreER^T2and ROSALUC as controls are indicated. (b) BLI image of animals induced with Tam. Image was acquired 5 days after the last Tam feed. Color bar indicates photons/cm²/s/steradian with the minimum and maximum threshold value. (B) Monitoring luciferase expression in the different organs isolated from ROSALUC X Alb-CreERT² mice. The 4–8-weeks-old bitransgenic ROSALUC X Alb-CreERT² mice (induced and uninduced) were sacrificed and various organs were isolated. Tissue lysates obtained were subjected to a luciferase assay. Figure depicts induced and non-induced ROSALUC X Alb-CreERT². Hash sign indicates values <1 RLU/μg of total protein. Brain tissue sample from induced and non-induced mice showed values <1 RLU/μg of total protein and is not shown in the figure. Values above dashed line are considered as luciferase expression. Tissues from control single transgenic ROSALUC and Alb-CreERT² mice showed an average of <2 RLU/ μg of total protein and are not depicted in the figure. Number of mice analyzed for each group = 5. Student’s t-test, comparing values to induced liver results in **P < 0.01.

Figure 6.

In vitro coculture assay to evaluate Cre-activatable OVA expression in hepatocytes from ROSAOVA X Alb-CreER^T2 mice. The above figure depicts the OVA antigen cassette as integrated in the ROSA26 locus in ROSAOVA mice via RMCE. ROSAOVA mice were mated to Alb-CreER^T2. Eight-weeks-old double transgenic mice were used to evaluate activation of OVA expression. Hepatocytes were isolated from six male double transgenic ROSALUC X Alb-CreER^T2 mice of which three were induced with Tam (mice Nr 1–3) and three non-induced (mice Nr 4–6); these cells were cocultured with OT-I CD8+ T cells. As negative controls single transgenic Alb-CreER^T2 mice (Nr 7–9) and ROSAOVA mice Nr (10–12) mice were used. Activation of OT-I T cells was monitored via IFN-γ cytokine secretion in the supernatants by conventional ELISA.

Figure 7.

Induced hepatitis upon Tam treatment in OT-I X ROSAOVA X Alb-CreER^T2 mice. (A) Determination of ALT activity in blood of OT-I X ROSAOVA X Alb-CreER^T2 mice. The 8-weeks-old OT-I X ROSAOVA X Alb-CreER^T2 were induced with Tam and blood was collected for ALT analysis at Day 2. Number of mice analyzed for each group = 4. **P < 0.01 (student’s t-test). (B) Histology of mouse liver tissue. Paraffin embedded liver tissue section was stained with H&E. Liver histology of OT-I X ROSAOVA X Alb-CreER^T2 (a and d), control OT-I X ROSAOVA (b and e) and C57BL/6 (c and f) is shown with different magnifications ×100 (a–c) and ×200 (d–f). Arrow indicates mononuclear cell infiltration.