Tetracycline-controlled transgene activation using the ROSA26-iM2-GFP knock-in mouse strain permits GFP monitoring of DOX-regulated transgene-expression

Abstract

BackgroundConditional gene activation is an efficient strategy for studying gene function in genetically modified animals. Among the presently available gene switches, the tetracycline-regulated system has attracted considerable interest because of its unique potential for reversible and adjustable gene regulation.ResultsTo investigate whether the ubiquitously expressed Gt(ROSA)26Sor locus enables uniform DOX-controlled gene expression, we inserted the improved tetracycline-regulated transcription activator iM2 together with an iM2 dependent GFP gene into the Gt(ROSA)26Sor locus, using gene targeting to generate ROSA26-iM2-GFP (R26t1Δ) mice. Despite the presence of ROSA26 promoter driven iM2, R26t1Δ mice showed very sparse DOX-activated expression of different iM2-responsive reporter genes in the brain, mosaic expression in peripheral tissues and more prominent expression in erythroid, myeloid and lymphoid lineages, in hematopoietic stem and progenitor cells and in olfactory neurons.ConclusionsThe finding that gene regulation by the DOX-activated transcriptional factor iM2 in the Gt(ROSA)26Sor locus has its limitations is of importance for future experimental strategies involving transgene activation from the endogenous ROSA26 promoter. Furthermore, our ROSA26-iM2 knock-in mouse model (R26t1Δ) represents a useful tool for implementing gene function in vivo especially under circumstances requiring the side-by-side comparison of gene manipulated and wild type cells. Since the ROSA26-iM2 mouse allows mosaic gene activation in peripheral tissues and haematopoietic cells, this model will be very useful for uncovering previously unknown or unsuspected phenotypes.

Tissue Distribution and Regulation of the Small Sar1b GTPase in Mice

“…In line with our results showing disparate enrichment between organs, various reports have emphasized that various TG mouse models equipped, genetically manipulated in different genes expressed variable amounts of mRNA in peripheral tissues [40]. More specifically, the pCAG promoter in the Rosa26 locus suffers from mosaic transgene expression among organs [41] and the responder gene expression was moderate and highly variable between and within different tissues [42]. …”

Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML 1‐ ETO mouse model

“…To completely restrict AE expression to haematopoietic cells, we transplanted whole bone marrow (BM) from compound ROSA26-iM2-tetO-GFP/TgPtet-AML1-ETO (R26/AE) mice into lethally irradiated recipients (Rhoades et al, 2000 ; Wortge et al, 2010 ). As illustrated in Fig 1 A, in reconstituted mice the expression of AE and the GFP co-reporter gene can be regulated by administering doxycycline (DOX) [for review see (Bockamp et al, 2002 ; Bockamp et al, 2008 )].…”

“…As illustrated in Fig 1 A, in reconstituted mice the expression of AE and the GFP co-reporter gene can be regulated by administering doxycycline (DOX) [for review see (Bockamp et al, 2002 ; Bockamp et al, 2008 )]. We have previously shown that the R26-iM2 effector mouse strain directed mosaic transgene expression to lineage − /c-Kit + /Sca1 + (L − K + S + ) haematopoietic progenitor cells and to different adult blood lineages (Wortge et al, 2010 ). In line with this mosaic expression pattern, we found conditional GFP activation in a percentage of long-term repopulating haematopoietic stem cells (LT-HSC, L − K + S + CD150 + CD48 − Flt3 − CD34 − ), short-term repopulating haematopoietic stem cells (ST-HSC, L − K + S + CD150 + CD48 − Flt3 − CD34 + ), common myeloid progenitors (CMPs, L − K + S − IL7Rα − CD34 + FcγRII/III low ), granulocyte macrophage progenitors (GMPs, L − K + S − IL7Rα − CD34 + FcγRII/III + ), megakaryocyte erythrocyte progenitors (MEPs, L − K + S − IL7Rα − CD34 − FcγRII/III low ) and common lymphoid progenitors (CLPs, LK int S int IL7Rα + ) of R26/AE reconstituted recipients ( Fig 1 B, Supporting Information Fig S1A).…”

“…As illustrated in Fig 1A, in reconstituted mice the expression of AE and the GFP co-reporter gene can be regulated by administering doxycycline (DOX) [for review see (Bockamp et al, 2002;Bockamp et al, 2008)]. We have previously shown that the R26-iM2 effector mouse strain directed mosaic transgene expression to lineage À /c-Kit þ / Sca1 þ (L À K þ S þ ) haematopoietic progenitor cells and to different adult blood lineages (Wortge et al, 2010). In line with this mosaic expression pattern, we found conditional GFP activation in a percentage of long-term repopulating haematopoietic stem cells (LT-HSC, L À K þ S þ CD150 þ CD48 À Flt3 À CD34 À ), short-term repopulating haematopoietic stem cells (ST-HSC,…”

Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation

“…Although this locus has been described to be ubiquitously active ( 32 ), expression of transgene cassettes with different transcription control elements underlies non-predictable expression characteristics ( 52 , 53 ). While Tet promoter driven transgene expression could be achieved to a certain degree, mosaic and also loss of expression have been reported ( 34 , 54 – 56 ). This suggests that the Tet promoter is affected by epigenetic silencing, although the underlying mechanism remained elusive.…”