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Tetracycline-controlled transgenic targeting from the SCL locus directs conditionalexpression to erythrocytes, megakaryocytes, granulocytes, and c-kit–expressinglineage-negative hematopoietic cellsErnesto Bockamp, Cecilia Antunes, Marko Maringer, Rosario Heck, Katrin Presser, Sven Beilke, Svetlana Ohngemach, Rudiger Alt,Michael Cross, Rolf Sprengel, Udo Hartwig, Bernd Kaina, Steffen Schmitt, and Leonid Eshkind The stem cell leukemia gene SCL, also
gene expression was restricted to erythro-
data therefore demonstrate that exog-
known as TAL-1, encodes a basic helix-loop-
cytes, megakaryocytes, granulocytes, and,
enously inducible and reversible expres-
helix transcription factor expressed in ery-
importantly, to the c-kit–expressing and lin-
sion of selected transgenes in myeloid,
throid, myeloid, megakaryocytic, and hema-
eage-negative cell fraction of the bone mar-
megakaryocytic, erythroid, and c-kit–ex-
topoietic stem cells. To be able to make use
row. In addition, conditional transgene acti-
pressing lineage-negative bone marrow cells
of the unique tissue-restricted and spatio-
vation also was detected in a very minor
can be directed through SCL regulatory
temporal expression pattern of the SCL
population of endothelial cells and in the
elements. The SCL knock-in mouse pre-
gene, we have generated a knock-in mouse
kidney. However, no activation of the re-
sented here represents a powerful tool for
line containing the tTA-2S tetracycline trans-
porter transgene was found in the brain of
studying normal and malignant hematopoi-
activator under the control of SCL regula-
adult mice. These findings suggested that
esis in vivo. (Blood. 2006;108:1533-1541)
tory elements. Analysis of this mouse using
the expression of tetracycline-responsive
reporter genes recapitulated the known en-
strains demonstrated that switchable trans-
dogenous expression pattern of SCL. Our
2006 by The American Society of Hematology
The basic helix-loop-helix transcription factor stem cell leukemia (SCL) endogenous SCL expression pattern.18-24 Complementary studies (also known as TAL-1 or TCL5) was originally identified by virtue of a examining the expression of a lacZ reporter knocked into exon III chromosomal translocation associated with acute human lymphoblastic of the SCL gene locus provided evidence that SCL regulatory leukemia.1-3 In addition to its involvement in leukemia, loss-of-function elements can direct expression of the lacZ transgene to progenitors studies in mice demonstrated an essential role of SCL for the specifica- of lymphoid, erythroid, and myeloid lineages.25 Analysis of SCL tion of mesoderm to primitive and definitive blood cell formation lacZ knock-in embryos further revealed expression of the reporter (reviewed in Begley and Green4 and Lecuyer and Hoang5). The absolute gene in parts of the central nervous system, the vascular endothe- requirement for SCL expression during early embryonic development lium, and in primitive and definitive blood cells.26 These findings, has led to the view that SCL acts as a master regulator of blood cell together with the loss-of-function data, suggest that SCL regulatory formation.6 Furthermore, conditional gene targeting of SCL in adult elements are active in HSCs and blood progenitors and that this mice not only has revealed a regulatory function of SCL in both activity is selectively maintained during ontogeny in myeloid, erythropoiesis and megakaryopoiesis,7-9 but also has suggested that SCL erythroid, megakaryocytic, and HSCs/progenitors but extinguished function is not required for self-renewal or long-term repopulation in all other mature blood cell lineages.
capacity of hematopoietic stem cells (HSCs). Within blood cell lineages, To be able to reversibly express transgenes in SCL-positive SCL expression has been reported in granulocytic, erythroid, megakaryo- blood cells, we have made use of the tetracycline regulatory cytic, and HSC/progenitor populations.4,5 system.27 Tetracycline-mediated control of transgenes has become Human and murine SCL genes are transcribed from 3 distinct an excellent strategy for studying gene function in mice (Gossen lineage-specific promoters leading to a complex pattern of differen- and Bujard28 and Bockamp et al29). Since transgene expression in tially spliced transcripts.10-16 DNase I hypersensitivity mapping, these animals is exclusively dependent on the administration/ restriction endonuclease accessibility assays, and functional in absence of tetracycline or tetracycline derivatives,30 the function of vitro experiments revealed several enhancer and silencer elements any gene product can be studied during selected developmental within the SCL genomic locus.17 In addition, reporter mice were windows or at critical stages of disease. Furthermore, inducible used to identify distinct regulatory elements of the SCL locus expression of toxic genes can be used to ablate selected cell responsible for directing expression to specific subdomains of the populations in vivo, allowing direct studies of the function of the From the Institute of Toxicology/Mouse Genetics and the Department of berg-Universita¨t Mainz (E.B.), and the Deutsche Krebshilfe (E.B.).
Hematology/Oncology, University Medical School, and the FACS and Array E.B. and C.A. contributed equally to this work.
Core Facility, Johannes Gutenberg-Universita¨t Mainz; the Department ofHematology/Oncology, University of Leipzig; and the Max-Planck-Institute for Reprints: Ernesto Bockamp, Institute of Toxicology/Mouse Genetics,
Medical Research, Heidelberg, Germany.
Johannes Gutenberg-Universita¨t Mainz, Obere Zahlbacher Str 67, 55131Mainz, Germany; e-mail: [email protected].
Submitted December 12, 2005; accepted April 21, 2006. Prepublished online asBlood First Edition Paper, May 4, 2006; DOI 10.1182/blood-2005-12-012104.
The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is hereby Supported by the European Union (E.B.), the Deutsche Forschungsge- marked ‘‘advertisement'' in accordance with 18 U.S.C. section 1734.
meinschaft (E.B., L.E.), the Stiftung Rheinland-Pfalz fu¨r Innovation (E.B.),the Mainz-Forschungsfonds (MAIFOR) program from the Johannes Guten- 2006 by The American Society of Hematology BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5 BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5 targeted cells and the creation of conditional disease models.31 The blotting using an 800-bp fragment upstream of SCL exon Ia as a 5⬘ outside unique experimental potential of tet on/off mouse models for probe and a 1025-bp polymerase chain reaction (PCR) fragment as an approaching crucial questions about normal and malignant blood inside probe to confirm correct integration. The 800-bp 5⬘ probe was cell development is illustrated by numerous reports investigating excised by Hind III digestion of the ⫺2000 SCL Ia pGL-2 plasmid,13 andthe 3⬘ probe was generated by PCR using oligonucleotide 5⬘-CCTCA- the in vivo function of conditionally expressed transgenes.32-41 In GAAGCTGTCACTGTGTC-3⬘ as a forward and oligonucleotide 5⬘- these reports, the combination of a tissue-specific effector with a TTGCTCAGGGACTTTACTGTCAG-3⬘ as a reverse primer. For in vivo responder mouse was used to express selected genes in a tetracy- excision of the neomycin-resistant cassette, germ-line–transmitting SCL- TA-2S knock-in mice were crossed to the SYCP-Cre deleter line.49 For studying the etiology of hematologic malignancies and, in Successful excision of the cassette was confirmed by using a 3-primer PCR particular, leukemias, the ability to control gene function in vivo is approach with the oligonucleotides 5⬘-TGGCCAAGTTACTCAATGACC-3⬘ a major advantage, since reversible induction can reveal whether and 5⬘-GGAAGTATCAGCTCGACCAA-3⬘ as forward primers and the transgene expression is needed for initiation, progression, mainte- 5⬘-GGATGGATCAACATGGACCT-3⬘ oligonucleotide as reverse primer.
nance, or remission of the disease. In addition, for several The LC-1, the enhanced green fluorescent protein (EGFP)–lacZ, and the leukemias, distinct oncogenes or leukemia-associated factors have tetO-Cre tetracycline-responsive responder lines have been described.50-52 been reported to be already expressed in HSCs or blood cell Genotyping of mice
progenitors.42,43 This observation, together with the obvious similar-ity between stem cells and cancer cells, has led to the emerging For genotyping of the SCL-tTA-2S knock-in mouse primers 5⬘- concept of the leukemic stem cell.44,45 Research focusing on the role of leukemic stem cells would therefore greatly benefit from GCTCC-3⬘ were used. The LC-1 mouse was typed using primers mouse models allowing the reversible induction of oncogenes and/or leukemia-associated factors in HSCs or blood cell GTTCTGCGGG-3⬘. The EGFP-lacZ tetracycline-responsive respondermouse was typed using primers 5⬘-CTCAAGTTCATCTGCACCACC-3⬘ To be able to reversibly target the expression of transgenes to SCL-positive cells, we have generated an SCL tTA-2S knock-in mouse. Detailed analysis of this mouse demonstrated that inhematopoietic tissues tetracycline-mediated transgene expression Organs from adult mice were dissected, extracted, and assayed for was completely restricted to myeloid, megakaryocytic, and ery- luciferase activity as described.53 Luciferase activity was normalizedagainst the amount of 10 ␮g protein. A linear relationship between light throid cells, and, most importantly, to c-kit–expressing lineage- units and volume was confirmed in all experiments. Luciferase values in the negative cells of the bone marrow. In addition, conditional presence and without doxycycline (DOX) were obtained in each case from transgene expression also was found in a very minor fraction of at least 3 different animals producing a similar pattern of activity.
platelet endothelial cell adhesion molecule 1 (PECAM-1)–expressing endothelial cells and in a subset of cells in the kidney.
However, no induction of transgenes was detected in histologicbrain sections. These findings suggest that the SCL tTA-2S Dissected tissues were digested at 37°C for 40 minutes in phosphatebuffered saline (PBS) (pH 7.4) containing 0.5 ␮g/mL collagenase together knock-in mouse recapitulates the known endogenous expression with 50 units DNase I per mL (both Sigma, St Louis, MO) and subsequently pattern of SCL. The SCL knock-in mouse presented here therefore subjected to fluorescence activated cell sorting (FACS) analysis.
represents an excellent model for studying controlled gene expres-sion in SCL-positive blood cells and, most importantly, to condition- FACS analysis and cell sorting
ally direct expression of selected gene products to c-kit⫹/lin⫺hematopoietic cells of the bone marrow.
Lineage contribution of EGFP-marked blood cells was analyzed with a4-color–equipped FACSCalibur (Becton Dickinson [BD], San Jose, CA) byco-staining with phycoerythrin (PE)–conjugated antibodies against CD11b,CD19, Gr-1, TER119 (BD), CD3, CD11c, DX5 (Caltag, Burlingame, CA), Materials and methods
CD23 (Southern Biotech, Birmingham, AL) or with purified antibodiesagainst CD41 (BD) detected with anti–rat-PE (Caltag). Collagenase-treated Construction of the targeting vector
suspensions of peripheral organs were simultaneously incubated with an The murine genomic SCL locus was obtained by screening a 129/Sv lambda endothelial-specific PECAM-1 rat monoclonal antibody (CD31, BD) and a phage library. A 4.2-kb fragment upstream of SCL exon V was used as the 5⬘ mix of TER119/CD45 antibodies (BD). Prior to staining, the samples (not homology arm and an 8.1-kb fragment downstream of the unique XbaI site the samples stained with secondary reagents) were blocked with PBS in exon VI as the 3⬘ homology arm and cloned into pGem11 ZF⫹ (Promega, supplemented with 5% rat serum for 10 minutes. Dead cells were excluded Madison, WI). All ATG codons of exon IV and the first ATG codon in exon from analysis via 7AAD staining (BD). Detection levels over background V were changed to GGG codons, thus preventing translational initiation were confirmed for the PECAM-1 antibody in parallel control experiments from these sites. The unique Not I recognition site in exon V was used for using a rat PE-conjugated IgG 2A isotype control antibody (BD). The stem insertion of the tTA-2S transactivator,46 followed by the bovine growth cell fraction was defined by lin⫺PE⫺ and c-kit⫹APC (CD117, BD) staining.
hormone polyA signal and a loxP-flanked neomycin-resistant cassette under Data were analyzed using the CellQuest Pro software (BD). In all cases the the control of the Herpes simples virus TK promoter (Figure 1A). All lineage contribution of EGFP-expressing cells was determined in 3 modified sequences were confirmed by sequence analysis.
independent experiments, analyzing each time a minimum of 5 ⫻ 105 cells.
Preparative FACS sorting of lin⫺ c-kit⫹ cells was performed using a FACS Vantage SE Turbo (BD). Lin⫹ cells were first depleted from the femoral mononuclear population using a magnetic affinity lineage depletion The W9.5 embryonic stem (ES) cell line47 was electroporated with the kit (MACS, Miltenyi Biotech, Auburn, CA). The lineage-depleted fraction linearized targeting vector. G-418–resistant single clones containing the was then stained with c-kit-APC antibody and the c-kit⫹ population sorted correctly recombined locus were injected into blastocysts and transferred simultaneously into EGFP⫹ and EGFP⫺ fractions. Because of the small into pseudopregnant mothers following standard procedures.48 Successful number of lin⫺ c-kit⫹ cells available, the EGFP sort gates were preset using germ-line transmission and correct integration was confirmed by Southern mononuclear cells from DOX-treated and untreated mice.

BLOOD, 1 SEPTEMBER 2006 INDUCIBLE EXPRESSION FROM THE SCL LOCUS 䡠 VOLUME 108, NUMBER 5 Figure 1. Targeting strategy and confirmation of the recombined SCL genomic locus. (A) Schematic overview of the targeting strategy. In the upper representation the
SCL wild-type genomic locus is shown. Coding exons (IV, V, and VI) are depicted as black, and noncoding exons (Ia, Ib, IIb, III, and part of VI) are depicted as white boxes. The
targeting construct is shown below the SCL genomic locus, consisting of 2 homology arms, the tTA-2S coding sequence (striped box), and the floxed neomycin-resistant
selection cassette (gray box). In the targeting construct all ATG codons in exon IV and the first ATG in exon V were changed to GGG codons. LoxP Cre-recombinase recognition
sites flanking the neomycin cassette are indicated as black triangles. Below the targeting construct the recombined mutant SCL locus is shown still containing the neomycin
cassette (Neo⫹). At the bottom of the representation the recombined SCL locus is depicted after excision of the neomycin cassette (Neo⫺). H indicates Hind III; R, EcoRI; N, Not
I; X, XbaI; A, ApaI, and B, BamHI. (B) 5⬘ confirmation of the recombined SCL locus by Southern blotting using a specific outside probe. Digestion with Hind III of wild-type (WT)
DNA gives rise to a 13-kb fragment, whereas the correctly recombined locus will result in a smaller 11.2-kb fragment (GT1 and GT2, germ-line–transmitting mouse founder line
1 and 2). (C) 3⬘ confirmation of the recombined SCL locus by Southern blotting. BamHI digestion of genomic DNA followed by hybridization with an inside probe produces a
4.9-kb fragment for the wild-type allele (WT) and a 2.4-kb fragment for the mutant knock-in allele (GT1). (D) In vivo excision of the neomycin-resistant cassette. PCR was used
to verify the excision of the neomycin-resistant cassette from the germ-line of the SCL tTA-2S knock-in mouse. The recombined SCL locus still containing the cassette will
produce a 1491-bp amplification product (Neo⫹). After excision of the neomycin cassette the same primers will amplify a 242-bp fragment (Neo⫺). The 764-bp amplification
product is specific for the SCL wild-type allele.
CAFC assay
(Perkin Elmer Life Sciences, Shelton, CT). Images were captured using acolor view digital camera running on an Olympus BX50 WI microscope The cobblestone area-forming cell (CAFC) assay was performed essentially (Olympus, Hamburg, Germany) and a 20⫻/0.50 numeric aperture objec- as described.54,55 Briefly, the lin⫺ c-kit⫹ EGFP⫹, lin⫺ c-kit⫹ EGFP⫺, and tive. Images were captured using a Color View 12 digital charge-coupled the whole mononuclear cell populations were counted, then titrated through device (CCD) camera (Olympus). Images were digitalized using the serial dilutions onto established OP-9 stromal feeder layers, each cell analySIS software package 3.1 (Soft Image Systems, Mu¨nster, Germany) concentration being represented by 20 independent wells. Cultures were fed and imported into Adobe Photoshop 4.0 (Adobe Systems, San Jose, CA). In by refreshing half of the medium weekly. All wells were scored for the all cases, electronic adjustments were applied to the whole image.
presence of cobblestone areas (groups of 5 or more hematopoietic cells ␤-Galactosidase expression and Cre expression in the brains of mice growing underneath the stromal layer) at day 14 and day 35 of culture, and were analyzed as described.51 the frequency of CAFCs calculated using Poisson statistics.
Controlled expression of transgenes
To exogenously switch the expression of luciferase, EGFP, and ␤-galactosi- dase in tTA-2S-SCL/LC-1 or tTA-2S-SCL/EGFP-lacZ tetracycline-responsive mice, animals were either provided with normal drinking water Generation of the SCL tTA-2S knock-in mouse
(reporter gene expression on) or fed a solution of 7.5 mg DOX (Sigma)/mL To conditionally express transgenes under the control of SCL water containing 1% sucrose (reporter gene expression off).
regulatory elements, gene targeting was used to insert the coding Immunofluorescence and X-gal staining
sequence for the tTA-2S transactivator46 into exon V of the SCLgene locus. We selected insertion of tTA-2S into exon V to ensure Mice were killed by cervical neck dislocation and organs snap frozen in that all known SCL regulatory elements were present in the isopenthane. Cryostat sections (5-12 ␮m) were fixed in 100% acetone at recombined locus.12-23,56,57 Figure 1A shows a schematic represen- 4°C for 1 hour, air dried, and stained for ␤-galactosidase by washing twice tation of the targeting strategy. Correct homologous recombination in PBS (pH 7.4), followed by overnight incubation at 37°C in X-gal in ES cells and germ-line transmission was confirmed by Southern solution (5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 1 mg/mLX-gal in PBS). To visualize endothelial cells, sections were incubated with a blotting (Figure 1B,C). Consistent with the introduction of 2 novel purified rat anti–mouse CD31 monoclonal antibody against PECAM-1 Hind III sites in the recombined locus, an 11.2-kb band was (BD), followed by a second biotin-conjugated goat anti–rat Ig–specific detected in addition to the 13-kb wild-type band after digestion of polyclonal antibody (BD) using the Renaissance TSA fluorescence system genomic DNA from the germ-line–transmitting founder animals

BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5 and hybridization with the 5⬘ outside probe (Figure 1B). Similarly, will bind to the tetO sequence upstream of the cytomegalovirus correct 3⬘ recombination was confirmed by BamHI digestion of (CMV) minimal promoter, resulting in transcriptional activation of genomic DNA, followed by Southern hybridization with an inside the luciferase transgene.
probe. As shown in Figure 1C in the germ-line–transmitting SCL expression in the adult is mainly restricted to hematopoi- founder GT1, the expected 2.4 kb was detected in addition to the etic tissues.4,5 In addition, the presence of a small number of 4.9-kb wild-type specific band (see also the schematic representa- SCL-positive cells also has been reported for the adult kidney.58 To tion of the expected fragments in Figure 1A). Correct recombina- evaluate if the SCL-tTA-2S effector mouse also will direct condi- tion was further confirmed for the overlap between the 3⬘ targeting tional expression of transgenes to these cells, SCL-tTA-2S knock-in arm and the adjacent genomic SCL locus using 2 additional probes effector mice were crossed to the LC-1 reporter mouse line.50 In (data not shown). Taken together, Southern blot analysis of the this mouse the luciferase gene is under the control of a tetracycline- germ-line–transmitting founder GT1 demonstrated correct homologous responsive promoter element. As expected, extracts prepared from recombination into the SCL locus.
different organs of bitransgenic SCL-tTA-2S/LC-1 mice, kept in the To completely exclude unwanted transcriptional interference presence of DOX, did not show luciferase activity (bottom bar effects from the TK promoter governing the expression of the graph ⫹DOX in Figure 2B, luciferase off). By contrast, high levels neomycin-resistant cassette, this cassette was removed from the of luciferase activity were detected in bone marrow and spleen of recombined SCL locus by in vivo excision using the SYCP-Cre- bitransgenic littermates that were never exposed to DOX (upper bar deleter mouse line.49 Successful excision of the floxed neomycin- graph ⫺DOX in Figure 2B, luciferase on). In addition, lower resistant cassette was confirmed by PCR. As shown in Figure 1D, luciferase activity was found in the thymus of induced animals.
removal of the floxed cassette resulted in a 242-bp PCR product Interestingly, extracts prepared from brain, heart, kidney, liver, (lane Neo⫺). By contrast, the recombined locus still containing the lung, tongue, esophagus, and pancreas also exhibited luciferase neomycin-resistant cassette produced a 1491-bp PCR product (lane activity over background, suggesting the presence of tTA-2S– Neo⫹). A 764-bp product specific for the wild-type SCL locus was expressing cells in these tissues. No substantial luciferase activity detected both in wild-type (lane WT) and rearranged mice (lanes was detectable in the salivary gland, the stomach, the small and Neo⫹ and Neo⫺), indicating the presence of at least one SCL large intestine, the muscle, or the lymph nodes. These results wild-type allele. For all subsequent experiments heterozygous demonstrated that the SCL-tTA-2S effector mouse induced reporter SCL-tTA-2S mice lacking the neomycin-resistant cassette were gene activity in adult hematopoietic tissues and that this expression used (homozygous SCL-tTA-2S knock-in mice were embryonic was strictly dependent on DOX (compare luciferase activity lethal, data not shown).
between bitransgenic mice in the presence and absence of DOX inFigure 2B). The observed high levels of luciferase activity in bone Tissue-specific expression of transgenes with the SCL tTA-2S
marrow and spleen were expected, as SCL is known to be expressed knock-in mouse is completely dependent on DOX
in these tissues. The low luciferase activity in the thymus is The schematic representation in Figure 2A illustrates the DOX- probably explained by the presence of a minor population of dependent regulatory strategy used here. As shown in Figure 2A, in CD8/CD4 double-negative and/or positive thymocytes or other the presence of DOX the tTA-2S transactivator does not bind to the cells of hematopoietic origin. Whether the somewhat unexpected tetO binding sequence and, thus, transgene expression is not luciferase activity in brain, heart, liver, lung, tongue, esophagus, initiated. Conversely, in the absence of DOX tTA-2S homodimers and pancreas represented organ-specific activation of the reporter Figure 2. Tissue-specific induction of the luciferase transgene is completely DOX-dependent. (A) Schematic representation of the tetracycline regulatory system.
Restriction endonuclease recognition sites are as in Figure 1. DOX indicates doxycycline; tTA-2S, tetracycline-dependent transactivator; tetO, DNA-binding consensus for
tTA-2S homodimers; pCMV, human cytomegalovirus minimal promoter; pA, polyA signal. (B) Luciferase activity expressed as relative light units (RLU) per microgram of protein
extract was determined for different organs as indicated. The top bar graph shows luciferase activities of double heterozygous SCL-tTA-2S/LC-1 mice in the absence of DOX
(⫺DOX, luciferase on). The bottom bar graph represents luciferase values obtained from double-transgenic SCL-tTA-2S/LC-1 mice that were kept from conception onwards in
the presence of DOX (⫹DOX, luciferase off). The luciferase values in each graph are shown for a single bitransgenic mouse. A similar pattern of activity was obtained also in 2
additional independent experiments using different mice.
BLOOD, 1 SEPTEMBER 2006 INDUCIBLE EXPRESSION FROM THE SCL LOCUS 䡠 VOLUME 108, NUMBER 5 gene or was the result of tTA-2S expressing circulating blood and that each individual cell can be simultaneously analyzed for the and/or endothelial cells could not be addressed at this point.
presence of several different tissue-specific markers. First, we Finally, the detected luciferase activity in the kidney was in line wanted to determine the overall percentage of transgene-expressing with the published expression of SCL in this organ.58 cells in lung, heart, kidney, tongue, and esophagus. The result ofthis analysis is shown in Figure 4 and demonstrated that lung, heart, Histologic and flow-cytometric analysis of transgene induction
kidney, tongue, and esophagus of noninduced bi-transgenic ani- in peripheral organs
mals did not contain any EGFP⫹ cells (data not shown). Consistent In the adult, SCL is restricted to hematopoietic cells and the with the previously detected luciferase activity, a small fraction of kidney.4,5,58 In addition, expression of endogenous SCL in endothe- EGFP-expressing cells was present in lung (1.8%), heart (1.71%), lial cells has been described for the early embryo, the vasculature of kidney (1.29%), tongue (0.67%), and esophagus (1.09%) of tumors, and the lining of newly arising blood vessels but is absent induced animals (Figure 4, ⫺DOX).
in quiescent adult vasculature.59-63 Intriguingly, lysates obtained To distinguish whether conditionally induced EGFP-expressing from SCL-tTA-2S/LC-1 mice exhibited luciferase activity in heart, cells were organ specific or represented migrating blood cells liver, lung, tongue, esophagus, and pancreas (Figure 2B). To and/or rare tTA-2S–expressing endothelial cells, EGFP⫹ cells were clarify, if transgene induction in the SCL-tTA-2S knock-in mouse tested for co-expression of the endothelial marker PECAM-1 was due to endogenous organ-specific expression or reflected the together with CD45 and TER119 pan-hematopoietic markers. The presence of circulating blood cells and/or resident endothelial cells, result of these experiments is shown in the central panel of Figure 4 SCL tTA-2S knock-in mice were mated to EGFP-lacZ tetracycline- and indicates that in lung, heart, esophagus, and tongue the responsive reporter mice.51 The resulting bitransgenic SCL tTA-2S/ majority of EGFP⫹ cells were of hematopoietic origin (CD45⫹/ EGFP-lacZ mice were either kept from conception onwards in the TER119⫹ cells contained in the 2 upper quadrants of each organ presence of DOX (reporter gene off) or kept on normal drinking plot). In the boxes on the right of Figure 4 the percentage of water (reporter gene on). At the age of 6 to 8 weeks organs from EGFP-expressing cells falling either into the category blood these mice were subjected to histologic analysis. As shown in the (CD45⫹/TER119⫹, large upper box) or endothelium (exclusively left panel of Figure 3, heart, liver, and kidney of bitransgenic SCL PECAM-1 expressing, bottom right box) and other cell types tTA-2S/EGFP-lacZ mice harbored blue ␤-galactosidase–express- (CD45⫺/TER119⫺ and PECAM-1⫺, bottom left box) is indicated ing cells, consistent with the previously detected luciferase activity for each organ. Even though a significant proportion of EGFP⫹ in these organs. No ␤-galactosidase activity was detected in cells of the kidney expressed hematopoietic markers (52.4%), a bitransgenic animals permanently kept in the presence of DOX major population of kidney cells lacked expression of both the (data not shown) or in muscle (Figure 3G). Immunofluorescence endothelial PECAM-1 marker and the pan-hematopoietic combina- analysis for the endothelial-specific PECAM-1 marker further tion of CD45/TER119 surface antigens (47.1%). The presence of a revealed that ␤-galactosidase–expressing cells typically did not significant population of EGPF-expressing cells lacking blood and colocalize with PECAM-1–positive endothelial populations (Fig- endothelial markers suggests that in renal tissues tTA-2S is ure 3, right panel). These results indicated that in the analyzed expressed in a kidney-specific fashion. This observation is in line organs transgene expression was in general not directed to endothe- with the preciously described presence of SCL-expressing cells in the kidney.58 Finally, in all analyzed peripheral organs very few To analyze transgene-expressing cells of different organs more EGFP⫹ cells exclusively expressed the PECAM-1 endothelial precisely, dissected tissues from induced and noninduced SCL-tTA- marker (bottom right quadrant of each plot). This suggested that 2S/EGFP-lacZ bitransgenic mice were treated with collagenase and conditional transgene expression also was directed to very rare the resulting cell suspensions examined by FACS. Major advan- endothelial cells. This finding was further supported by control tages of this strategy are that large numbers of cells can be tested experiments using an isotype antibody instead of PECAM-1. In Figure 3. DOX-induced expression of ␤-galactosidase in peripheral organs of SCL-tTA-2S/EGFP-lacZ double-transgenic mice does not generally colocalize to
vascular endothelium. Representative sections from (A) heart, (C) liver, (E) kidney, and (G) muscle of double-transgenic mice were analyzed for the presence of
␤-galactosidase–expressing cells (left panel). Vascular endothelium was identified by immunofluorescence using a monoclonal antibody against murine PECAM-1 (B, D, F,
and H, right panel). The location of ␤-galactosidase–expressing cells is indicated by arrows.

BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5 lymph nodes (0.13%). These results indicated that induction of theEGFP reporter gene in these mice was strictly dependent on DOXand that expression of EGFP occurred only in a subset of cells.
To investigate more precisely whether conditional induction of EGFP was tissue restricted to certain blood cell types or whether allhematopoietic lineages contained EGFP-expressing cells, distincthematopoietic cell types were analyzed for the presence of EGFP.
As shown in Figure 6, no EGFP-positive DX5⫹ NK-cells, CD3⫹T-lymphoid cells, a very minor fraction of CD19⫹ cells, no CD23⫹mature B cells, activated macrophages, eosinophils, and folliculardendritic cells were detected in hematopoietic organs of inducedbitransgenic mice. Indeed, as no EGFP⫹ cells expressed CD23, thevery minor fraction of CD19-expressing EGFP⫹ cells mightrepresent early myelomonocytic cells and/or immature B cells. Bycontrast, in the same animals EGFP⫹ cells were detected in Gr1⫹granulocytes, TER119⫹ erythrocytes, CD41⫹ megakaryocytes, andthe c-kit/lin⫺ fraction.
To further evaluate the presence of HSCs/progenitor cells within the EGFP-expressing c-kit⫹/lin⫺ population, limiting dilu-tion cobblestone area-forming cell (CAFC) assays were performed.
CAFC assays are providing a generally accepted in vitro readout ofboth primitive and progenitor HSCs in mice.54,55,64 Cobblestone Figure 4. Induction of EGFP in peripheral organs of SCL-tTA-2S/EGFP-lacZ
areas apparent after 14 days accurately measure spleen colony- double-transgenic mice is primarily restricted to hematopoietic cells and a
subset of organ-specific cells in the kidney. Representative FACS profiles of
forming units (CFU-S) day 12, and those present after 35 days of collagenase-digested tissues from lung, heart, kidney, tongue, and esophagus are culture contain long-term HSC repopulating activity.54,55,64 To shown. Left panel (⫺DOX): Induced organs of bitransgenic mice do contain a small investigate if the EGFP-expressing population of bone marrow fraction of EGFP⫹ cells (bottom right quadrant). Percentages of EGFP-expressingcells are shown in the top right quadrant. Central panel: The EGFP⫹ fraction of cells cells did contain CAFC activity, lin⫺/c-kit⫹ EGFP⫹, lin⫺/c-kit⫹ from the left panel of organ plots (indicated by an arrow) was used for plotting EGFP⫺, and, as a negative control, mononuclear bone marrow cells CD45/TER119 pan-hematopoietic markers (y-axis) against the PECAM-1 endothelial of induced SCL-tTA-2S/EGFP-lacZ mice were preparatively sorted marker (x-axis). Right panel: Percentages of EGFP⫹ hematopoietic cells are shownin the large top box and percentages of endothelial cells in the bottom right box. The and tested for their CAFC activities. As expected the mononuclear percentage of EGFP-expressing cells lacking blood and endothelial markers is fraction of bone marrow cells essentially contained no CAFCs indicated in bottom box on the left. Note the substantial increase of EGFP-expressing (Table 1, MNC). In contrast, day 14 and day 35 CAFCs were double-negative CD45⫺/TER119⫺and PECAM-1⫺ cells in the kidney.
several control experiments the absolute percentage of PECAM-1single-positive cells was in all cases higher than the percentagesdetected with the matched isotype antibody (Figure S2, available atthe Blood website; see the Supplemental Figures link at the top ofthe online article). For this reason we conclude that a very minorpopulation of all PECAM-1⫹ cells did express the tTA-2S transac-tivator. It is most likely that these cells represented newly formingor regenerating vasculature known to express SCL.59-63 In conclusion, our data suggest that in lung, heart, tongue, and esophagus, expression of tTA-2S was almost completely restrictedto hematopoietic cells. In the kidney the majority of EGFP-expressing cells were either hematopoietic or organ specific.
SCL regulatory elements target induction of EGFP to red blood
cells, megakaryocytes, granulocytes, and the c-kitⴙ/linⴚ
population of the bone marrow
Next, we wanted to determine in which hematopoietic lineages theSCL-tTA-2S effector mouse could induce expression of conditionaltransgenes. For this purpose reporter mice carrying the EGFPcoding region under the control of a tetracycline-inducible pro-moter51 were mated to the SCL-tTA-2S effector mouse line. In theresulting bitransgenic animals hematopoietic organs were analyzedfor the presence of EGFP⫹ cells by FACS. As shown in Figure 5,hematopoietic organs from bitransgenic effector/reporter mice Figure 5. Expression of EGFP in hematopoietic organs is dependent on DOX.
permanently kept in the presence of DOX did not contain any FACS analysis of adult spleen, bone marrow, thymus, and lymph nodes from EGFP⫹ cells (right panel ⫹DOX, EGFP off). By contrast, bi- double-transgenic effector/responder mice, demonstrating that the induction ofEGFP was strictly dependent on DOX. Note the lack of EGFP⫹ cells in the FACS plots transgenic mice without DOX contained a fraction of EGFP⫹ cells on the right where EGFP expression was inhibited by DOX. The percentage of in spleen (1.3%), bone marrow (1.72%), thymus (0.03%), and EGFP-positive cells in each organ is indicated in the top right quadrant.

BLOOD, 1 SEPTEMBER 2006 INDUCIBLE EXPRESSION FROM THE SCL LOCUS 䡠 VOLUME 108, NUMBER 5 generate a mouse line allowing reversible targeting of transgeneexpression to HSCs and blood progenitors. Such a conditional SCLeffector mouse would be an invaluable experimental tool forapproaching fundamental issues concerning normal and malignanthematopoiesis.
The basic helix-loop-helix transcription factor SCL is one of the very few genes known to be expressed both in embryonic and adultHSCs.4,5 This unique expression pattern suggests that SCL regula-tory elements could be used to direct conditional expression toHSCs and blood cell progenitors. Radomska and colleagues36 hadpreviously used the human CD34 locus to direct tetracycline-controlled expression of heterologous transgenes to HSCs and Figure 6. Induction of EGFP expression in SCL-tTA-2S/EGFP-lacZ double-
early progenitors. In this mouse inducible transgene expression was transgenic mice is restricted to granulocytes, red blood cells, megakaryocytes,
and c-kitⴙ/linⴚ cells of the bone marrow. The presence of EGFP⫹ cells in DX5⫹ NK
reported for endothelial and early blood cell progenitors. In a cells, Gr1⫹ myeloid cells, TER119⫹ red blood cells, CD3⫹ T-lymphoid cells, CD19⫹ similar fashion elements from the 3⬘ SCL enhancer were used to cells, CD41⫹ megakaryocytes, CD23 mature B cells, activated macrophages, direct DOX-inducible expression of transgenes to hematopoietic eosinophils, follicular dendritic cells, and the bone marrow lin⫺/c-kit⫹ population wasdetermined by FACS.
tissues and HSCs.41 However, in this study, only lung, intestine,and hematopoietic organs were analyzed for DOX-dependenttransgene induction. For this reason it is not clear to what extent generated from the lin⫺/c-kit⫹ EGFP-expressing fraction, indicat- conditional expression was exclusively restricted to hematopoietic ing the presence of progenitors/HSCs proficient to generate early tissues and the lung but was absent from other organs. Interestingly, and late CAFCs (Table1). Furthermore, the lin⫺/c-kit⫹ EGFP- when this effector mouse was used to express the BCR-ABL negative fraction also contained CAFC activity. The presence of oncogene, a chronic myeloid leukemia (CML)–like disease was CAFC activity in both the lin⫺/c-kit⫹ EGFP-expressing and induced.41 However, since overexpression of SCL under the control EGFP-negative fraction is not surprising, since SCL is not homoge- of the 3⬘ SCL enhancer led only to a partial rescue of the lethal SCL neously expressed in hematopoietic progenitors/HSCs.65,66 How- knock-out phenotype, it is to be assumed that the 3⬘ enhancer is not ever, the generation of day 14 and day 35 CAFC with the sufficient for recapitulating the endogenous SCL expression pat- EGFP-expressing lin⫺/c-kit⫹ fraction suggests that the SCL- tern.23 Here, we report the generation of a tTA-2S knock-in mouse tTA-2S knock-in mouse line directs expression of EGFP to a subset line that mirrors the known expression pattern of SCL in the adult.
Transcriptional regulation of the murine SCL gene has been Taken together, our results show that the SCL-tTA-2S knock-in extensively studied in vitro and in vivo.12-23,56 Based on this line exclusively targeted EGFP expression to a subset of hematopoi- information, we reasoned that inserting the tTA-2S coding se- etic lineages, namely, erythrocytes, megakaryocytes, granulocytes, quence into exon V of the SCL locus would ensure the conservation and also to c-kit⫹/lin⫺ bone marrow cells. These findings sug- of critical regulatory elements and result in a faithful recapitulation gest that conditional targeting of the EGFP transgene recapitu- of the endogenous SCL expression pattern by tTA-2S. The capacity lated the reported lineage-restricted expression pattern of SCL in and tissue specificity of the SCL-tTA-2S effector mouse line was adult blood.
tested using luciferase, lacZ, and EGFP tetracycline-dependentreporter mice. In a first series of experiments the LC-1 luciferase Analysis of transgene induction in the brain
responder line50 was used to determine in which organs the Expression of SCL has been reported in V2b interneurons of the expression of the luciferase transgene was induced. Since lucif- developing embryo.67-69 In addition, in a recent report it was shown erase is known to be a sensitive reporter, low levels of transgene that SCL plays a critical role for the initial specification of primitive induction should be detectable. These experiments demonstrated neural precursors to astrocytes.69 However, SCL mRNA is not high and strictly DOX-dependent transgene induction in bone expressed in the brain of postnatal mice.70 Using the EGFP-lacZ marrow and spleen and intermediate levels in brain, heart, kidney, and the tetO-Cre responder mouse lines,51,52 functional tTA-2S liver, lung, tongue, esophagus, pancreas, and thymus (Figure 2B).
activity could not be detected in coronal sections through the entire The intermediate induction of luciferase activity in these organs brain of induced SCL-tTA-2S mice. The lack of Cre-recombinase was somewhat unexpected, as SCL expression in the adult had been expression in SCL-tTA-2S/tetO-Cre mice (data not shown) and theabsence of detectable ␤-galactosidase activity in induced SCL-tTA- Table 1. EGFP-expressing c-kitⴙ/linⴚ cells from the bone marrow
2S/lacZ-EGFP mice (compare induced and noninduced sections in of induced SCL-tTA-2S/EGFP-lacZ mice contain early and late
Figure S1) indicated that tTA-2S expression in the brain was either absent or too low to drive the expression of the indicator Experiment 1, CAFCs
Experiment 2, CAFCs
transgenes. We conclude, therefore, that the SCL-tTA-2S effector per 104 cells
per 104 cells
Tested cell
mouse is not suitable for robust expression of transgenes in the adult brain.
lin⫺ c-kit⫹ EGFP⫹ lin⫺ c-kit⫹ EGFP⫺ Bone marrow cells were isolated and cultured on OP-9 cells for limiting dilution analysis of CAFC activity as described in "Materials and methods." Mean CAFC The aim of this study was to generate a conditional mouse model frequencies scored at day 14 and day 35 are shown for 2 independent experimentsusing, in total, 4 different mice. Numbers in parentheses indicate the range of the 95% that recapitulates the unique spatio-temporal and lineage-restricted confidence limit.
expression pattern of the SCL gene. In particular, we wished to MNCs indicates mononuclear cells.
BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5 reported only for hematopoietic tissues and the kidney.4,5,58 How- cells was determined. In a previously published report of mice ever, given that the analyzed organs were not perfused prior to harboring a lacZ reporter gene in exon III of the SCL locus, lacZ dissection, we could not exclude the possibility that the measured expression was confined to HSCs, blood cell progenitors, and red luciferase activities were due to tTA-2S–expressing, circulating blood cells.25 These findings contrast with the endogenous SCL blood, and/or resident endothelial cells. To address this question expression pattern and also with the induced transgene expression and to visualize transgene expressing cells in situ, sections from pattern observed here, which also included megakaryocytes and kidney, muscle, liver, and heart of induced bitransgenic SCL-tTA- granulocytes. However, the differences between these 2 SCL 2S/EGFP-lacZ mice were stained for ␤-galactosidase activity.
knock-in lines are most likely explained by differences in the Inspection of these sections revealed the presence of lacZ- design of the targeting strategy (lack of the third SCL promoter and expressing blue cells in kidney, liver, and heart, but not in the actively transcribing neomycin gene in the case of the lacZ muscle (Figure 3). Subsequent staining of these sections with the knock-in line). Most notably, the SCL-tTA-2S knock-in mouse PECAM-1 endothelial-specific marker further revealed no obvious directed expression of inducible transgenes to c-kit⫹/lin⫺ bone general co-localization of tTA-2S and PECAM-1–expressing cells marrow cells known to contain blood progenitors/HSCs. Further- (Figure 3, right panel). Therefore, the histologic analysis suggested more, measurement of CAFC frequencies from tTA-2S–targeted that tTA-2S was not expressed in the majority of endothelial cells EGFP-expressing c-kit⫹/lin⫺ bone marrow cells demonstrated the of these organs. To further clarify the origin of tTA-2S–expressing presence of day 14 CAFCs and day 35 CAFCs, which are an cells in peripheral organs and to permit analysis of large numbers of accepted in vitro correlate for CFU-S and bone marrow repopulat- individual cells, we used FACS. As the EGFP-lacZ responder mice ing stem cell activity (Table1). The ability of EGFP⫹/c-kit⫹/lin⫺ will simultaneously express EGFP and lacZ upon induction,51 cells from the bone marrow to generate day 35 CAFCs thus kidney, heart, lung, esophagus, and tongue tissues were subjected strongly suggests that the SCL-tTA-2S knock-in mouse is suitable to collagenase digestion followed by FACS analysis. These experi- for conditional expression of transgenes in adult HSCs/progenitors.
ments showed that all analyzed tissues contained a fraction of cells Taken together, our data show that the SCL-tTA-2S knock-in expressing EGFP, thus confirming the previously measured lucif- mouse model will direct conditional DOX-dependent expression of erase activities in these organs (Figure 4). In addition, examination transgenes within blood to erythrocytes, megakaryocytes, granulo- of EGFP-expressing cells using blood- and endothelial-specific cytes, and, most importantly, to c-kit⫹/lin⫺ cells of the bone markers revealed that the majority of the analyzed cells were of marrow. This expression profile therefore represents a recapitula- hematopoietic origin and that only a minor subset represented tion of the known endogenous SCL expression pattern. It is to be endothelial or other cell types that were not analyzed further.
expected that the mouse presented here will be a valuable tool for Moreover, the kidney contained a significant proportion of EGFP- raising fundamental questions about normal and malignant blood expressing cells lacking both blood and endothelial markers, cell development.
directly suggesting that these cells were organ-specific (Figure 4).
This finding is in line with a recent report showing the expressionof SCL in the kidney.58 To determine if adult brain tissues were targeted by the SCL-tTA-2S knock-in mouse, ␤-galactosidaseinduction of neuronal tissues also was determined in SCL-tTA-2S/ We thank H. Bujard for the LC-1 reporter mouse line and the EGFP-lacZ mice. No difference between induced and noninduced tTA-2S transactivator cDNA. In addition, we are grateful to J.
brain tissues was seen in these mice, demonstrating that SCL Mann, who gave us the W9.5 ES cell line. We also would like to regulatory elements did not direct transgene induction to the brain.
thank the animal technicians of the Mainz animal house for Taken together, histologic and flow cytometric analyses suggested excellent assistance and mouse care, and the Interdisciplinary that the observed induction of transgenes closely mirrored the Center for Clinical Research (IZKF) Core Unit of Fluorescence known expression pattern of SCL.
Technology in Leipzig for preparative cell sorting. Finally, we would Finally, the specificity of tTA-2S–mediated transgene expres- like to acknowledge Annette Herold for her excellent technical assis- sion in mature blood cells and c-kit–expressing lineage-negative tance in the preparation and analysis of brain sections.
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Tetracycline-controlled transgenic targeting from the
conditional expression to erythrocytes, megakaryocytes, granulocytes,
and c-kit-expressing lineage-negative hematopoietic cells

Ernesto Bockamp, Cecilia Antunes, Marko Maringer, Rosario Heck, Katrin Presser, Sven Beilke,
Svetlana Ohngemach, Rudiger Alt, Michael Cross, Rolf Sprengel, Udo Hartwig, Bernd Kaina, Steffen
Schmitt and Leonid Eshkind
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