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. Author manuscript; available in PMC: 2008 Feb 7.
Published in final edited form as: Genesis. 2005 Nov;43(3):129–135. doi: 10.1002/gene.20162

In Vivo Genetic Ablation by Cre-Mediated Expression of Diphtheria Toxin Fragment A

Anna Ivanova 1, Massimo Signore 1, Nadia Caro 1, Nicholas DE Greene 1, Andrew J Copp 1, Juan Pedro Martinez-Barbera 1,*
PMCID: PMC2233880  EMSID: UKMS1477  PMID: 16267821

Summary

We generated a ROSA26-eGFP-DTA mouse line by introducing an eGFP-DTA (enhanced green fluorescent protein - diphtheria toxin fragment A) cassette into the ROSA26 locus by homologous recombination in ES cells. This mouse expresses eGFP ubiquitously, but DTA expression is prevented by the presence of eGFP, a Neo cassette, and a strong transcriptional stop sequence. Mice carrying this construct are normal and fertile, indicating the absence of DTA expression. However, upon Cre-mediated excision of the floxed region DTA expression is activated, resulting in the specific ablation of Cre-expressing cells. As an example of this approach, we ablated Nkx2.5 and Wnt1-expressing cells by using the Nkx2.5-Cre and Wnt1-Cre mouse lines, respectively. We observed loss of the precise tissues in which Nkx2.5 and Wnt1 are expressed. Apart from being a general GFP reporter, the ROSA26-GFP-DTA mouse line should provide a useful resource for genetic ablation of specific groups of cells.

Keywords: Cre, loxP, diphtheria toxin, eGFP, Nkx2.5, Wnt1, genetic ablation, heart, midbrain

The role of individual cells in complex tissues or within the whole organism can be examined by specific deletion of appropriate lineages. For example, during embryogenesis specific groups of cells (signalling centres) control the fate of neighbouring cells by emanating diffusible molecules (Meinhardt, 1983; Martinez et al., 1991; Shimamura and Rubenstein, 1997; Placzek and Briscoe, 2005). Mechanical ablation of restricted embryonic regions has been useful in identifying these signalling centres and their role during development (Placzek et al., 1995; Shimamura and Rubenstein, 1997; Thomas and Beddington, 1996). However, these experiments require very precise surgery on the developing embryo, which can often be a difficult task. In adulthood the loss of small numbers of strategically placed cells may result in conditions such as Parkinson’s disease (Moore, 2005), Type I diabetes (Butler et al., 2003), and Hirschsprung’s disease (Amiel and Lyonnet, 2001). Thus, a versatile system to specifically ablate cells of any lineage during embryogenesis or in adulthood would be of substantial benefit for studies of development, as well as to model human diseases of various aetiologies.

With the aim of developing such a system, we generated a ROSA26-eGFP-DTA mouse line, which combines the use of enhanced green fluorescent protein (GFP), diphtheria toxin A subunit (DTA), and Cre recombinase. eGFP is a mutated version of GFP that displays enhanced fluorescence in mammalian cells in vivo and in vitro (Srinivas et al., 2001). Diphtheria toxin is secreted by pathogenic strains of Corynebacterium diphtheriae and is composed of two subunits, A and B. Subunit B is responsible for the internalisation of the toxin upon binding to its receptor. Once inside the cell, subunit A catalyses the inactivation of elongation factor 2, resulting in termination of protein synthesis and apoptosis of the target cell (Maxwell et al., 1986; Palmiter et al., 1987; Breitman et al., 1990; Harrison et al., 1991; Collier, 2001). The phage-derived Cre/loxP system has been successfully used in vitro and in vivo to conditionally inactivate numerous genes (Nagy, 2000). Cre recombinase recognises 34 basepair loxP DNA fragments and mediates excision of the DNA flanked by two loxP sites if they are placed in the same orientation.

We generated the ROSA26-eGFP-DTA mouse line by insertion of a conditionally expressed DTA construct into the ROSA26 locus by homologous recombination in ES cells (Fig. 1a). In this construct, eGFP is upstream of DTA and is constitutively expressed under control of the ROSA26 promoter. In contrast, DTA transcription is prevented by the presence of a floxed DNA region containing eGFP, a Neo cassette, and a triple SV40 polyadenylation signal (tpA). This strategy allows the conditional expression of DTA upon Cre-mediated excision of the floxed region, which leads to specific death of Cre-expressing cells. ROSA26eGFP-DTA/+ and ROSA26eGFP-DTA/eGFP-DTA mice were present in the expected proportions among offspring of heterozygous intercrosses (Table 1). Hence, it is unlikely that DTA expression is leaky from the ROSA26 locus, as no toxic effect was detectable in these animals. When observed under ultraviolet light, ES cell clones, embryos, and mice carrying the ROSA26-eGFP-DTA allele showed ubiquitous green fluorescence (Figs. 1g, 2d, 3b).

FIG. 1.

FIG. 1

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Targeting of the ROSA26 locus. a: From top to bottom, diagrams of: pBigT-invloxP plasmid; pROSA26PA plasmid containing genomic ROSA26 sequences and PGK-DTA for negative selection in ES cells; wildtype ROSA26 locus; targeted ROSA26 locus before and after Cre-mediated excision of the loxP-flanked DNA (eGFP, PGK-Neo cassette and a triple SV40 polyadenylation signal). b,c: Examples of mice genotyped by Southern blot (b) and PCR (c). For Southern blot, genomic DNA was digested with EcoRV and hybridized with the probe indicated in a. PCR genotyping was performed as described (see Materials and Methods) using the indicated primers (arrows in a). d,e: Brightfield photographs of wildtype (c) and targeted (d) ES cell colonies. f,g: Fluorescence photographs of the colonies in d and e. Note that the targeted ES cell colony expresses eGFP. Scale bars = 100 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com]

Table 1.

Frequency of Genotypes Among Offspring From ROSAeGFP-DTA/+ Heterozygous Intercrosses

Genetic backgrounda Total number of offspring Number of offspring of each genotypeb
+/+ +/- -/-
C57BL/6J 44 12 25 9
CD1 43 7 20 16
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a

ROSAeGFP-DTA/+ heterozygotes were backcrossed twice with C57BL/6J or CD1 mice.

b

Distribution of offspring among genotypes does not differ from the expected 1 (+/+): 2 (+/-): 1 (-/-) ratio on either C57BL/6J or CD1 background p > 0.05 in each case).

FIG. 2.

FIG. 2

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Lack of cardiac tissue in ROSA26eGFP-DTA/+;Nkx2.5Cre/+ compound heterozygous embryos. a: 9.0 dpc Nkx2.5Cre/+ embryo. b: ROSA26eGFP-DTA/+;Nkx2.5Cre/+ compound mutant lacking the heart (arrow). c,d: Fluorescence photographs of the embryos shown in a and b. Only the compound heterozygote expresses eGFP. e,f: Haematoxylin-eosin staining of transverse sections at the level of the heart in Nkx2.5Cre/+ (e) and ROSA26eGFP-DTA/+;Nkx2.5Cre/+ compound heterozygote (f) at 9.0 dpc. The heart is absent from the compound heterozygous embryo, and the anterior cardinal veins (arrowheads) and dorsal aorta (arrows) appear dilated. Foregut, neural tube, and cranial mesenchyme all appear morphologically normal. g-j: Whole-mount in situ hybridisation with alpha-cardiac actin (aCa) (g,h) and Cre (i,j) riboprobes on Nkx2.5Cre/+ (g,i) and ROSA26eGFP-DTA/+;Nkx2.5Cre/+ compound heterozygotes (h,j) at 9.0 dpc. These cardiac markers are not detectable in compound heterozygotes. Scale bars = 200 μm (a-d,g-j); 100 μm. (e,f) a, atrial chamber; cv, common ventricle; f, foregut. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com]

FIG. 3.

FIG. 3

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Ablation of the midbrain region in ROSA26eGFP-DTA/+;Wnt1-Cre compound embryos. a: Left to right: Wnt1-Cre, ROSA26eGFP-DTA/+, and ROSA26eGFP-DTA/+;Wnt1-Cre 10.5 dpc embryos. Only the ROSA26eGFP-DTA/+;Wnt1-Cre compound embryo shows defects in the anterior neural tube. Other regions of the embryo appear normal by gross morphology. b: Fluorescence photographs of embryos in (a). Only those containing the ROSA26-eGFP-DTA targeted allele express eGFP. c,d: Magnified views of the head of 9.0 dpc ROSA26eGFP-DTA/+ and ROSA26eGFP-DTA/+;Wnt1-Cre embryos. Optic (arrowhead) and otic (arrow) vesicles are well developed in the compound mutant, but there is a significant lack of brain tissue between these vesicles. Scale bars = 500 μm (a,b); 200 μm (c,d). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com]

To prove the principle that specific cell ablations can be achieved during mouse development, we crossed ROSA26-eGFP-DTA and Nkx2.5-Cre mice. Nkx2.5-Cre is a knockin of Cre into the Nkx2.5 locus (Moses et al., 2001). When crossed with the general ROSA26lacZ reporter mouse, lacZ expression is weak in the heart crescent at headfold stages, but is robust in the developing heart tube at early somite stages (Moses et al., 2001). In accordance with this expression pattern, we found that ROSA26eGFP-DTA/+;Nkx2.5Cre/+ compound heterozygous embryos showed a fully penetrant phenotype characterised by the absence of the heart at the 12-14 somite stage (n = 11) (Fig. 2a-f). All other embryonic regions were normal by morphological, histological, and in situ hybridisation analyses. This phenotype was not observed in littermate embryos with any other genotype: ROSA26eGFP-DTA/+ and Nkx2.5Cre/+ single mutants as well as wildtype embryos were all entirely normal. To further confirm the absence of the heart, we analysed the expression of specific heart markers such as Nkx2.5 and alpha-cardiac actin (Sassoon et al., 1988; Lints et al., 1993; Lemonnier and Buckingham, 2004). Neither of these markers was expressed in ROSA26eGFP-DTA/+; Nkx2.5Cre/+ compound heterozygous embryos (n = 9) (Fig. 2g,h and data not shown). Cre expression was restricted to the heart in Nkx2.5Cre/+ embryos, but was absent from ROSA26eGFP-DTA/+; Nkx2.5Cre/+ embryos (n = 5) (Fig. 2i,j). At 9.5 days postcoitum (dpc), ROSA26eGFP-DTA/+; Nkx2.5Cre/+ compound heterozygous embryos were very small, morphologically abnormal, and in some cases partially resorbed (data not shown), suggesting that a functional cardiovascular system is required for survival between 8.5-9.5 dpc during mouse embryogenesis (Copp, 1995).

We also crossed the ROSA26eGFP-DTA/+ mice with Wnt1-Cre transgenics (Jiang et al., 2000). Wnt1-Cre is a transgenic line that drives Cre expression in the prospective midbrain at presomitic stages. At later stages, Cre expression is restricted to the dorsal aspects of these brain regions and throughout the dorsal hindbrain and spinal cord (Echelard et al., 1994; Rowitch et al., 1998; Jiang et al., 2000). At 9.5 and 10.5 dpc, ROSA26eGFP-DTA/+; Wnt1-Cre compound embryos showed abnormal morphology of the brain region (n = 10) (Fig. 3a-d). The brain rostral to the otic vesicle was significantly smaller and the cranial neural tube was open dorsally (exencephaly). Forebrain tissue was distinguishable and optic cups developed bilaterally (Fig. 3C,D). The isthmus, a constriction of the neural tube at the boundary between the midbrain and hindbrain (MHB), developed normally in Wnt1-Cre, ROSA26eGFP-DTA/+, and wildtype embryos, whereas it was absent in ROSA26eGFP-DTA/+;Wnt1-Cre mutants (Fig. 3a and data not shown). No other regions of the embryos showed gross abnormalities at these stages. The brain phenotype observed in ROSA26eGFP-DTA/+;Wnt1-Cre is similar to that described in Wnt1 null mutant embryos (McMahon and Bradley, 1990; Thomas and Capecchi, 1990).

To characterise this phenotype further we used in situ hybridisation with neural markers specific for restricted regions of the neural tube (Fig. 4). The most anterior Wnt1 expression domain, which encompasses the midbrain and posterior forebrain, was much reduced in ROSA26eGFP-DTA/+;Wnt1-Cre compound embryos at 8.5 dpc (n = 6) (Fig. 4a,b, bracketed region). Weak Wnt1 expression was detected at the MHB and dorsal hindbrain in the compound mutant at this stage (Fig. 4a,b, arrowhead and black arrow, respectively). However, normal Wnt1 expression was detected in the dorsal neural tube at spinal cord levels (Fig. 4a,b, red arrow). Cre expression, which has been previously shown to recapitulate Wnt1 expression (Echelard et al., 1994; Rowitch et al., 1998; Jiang et al., 2000), was also absent from the midbrain and posterior forebrain of ROSA26eGFP-DTA/+;Wnt1-Cre compound embryos at 9.0 dpc, except for a small patch of cells ventrally within the MHB (n = 4) (Fig. 4c,d).

FIG. 4.

FIG. 4

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Whole-mount in situ hybridisation analysis of Wnt1-Cre and ROSA26eGFP-DTA/+;Wnt1-Cre embryos. a,b: Wnt1 expression rostral to midbrain-hindbrain boundary (MHB) (arrowheads) is absent in the ROSA26eGFP-DTA/+;Wnt1-Cre compound embryo (b) when compared with the control (a) at 8.5 dpc (i.e., region shown by bracket in a is absent in b). Wnt1 expression in the dorsal aspects of the hindbrain is reduced in intensity (black arrows), but is normal in the spinal cord (red arrows). c,d: Expression of Cre at 9.0 dpc. The lack of midbrain tissue rostral to the MHB (arrowhead) is particularly evident at this stage, as shown by the significant reduction of Cre expression in the MHB (arrowhead) and rostral to it (bracket in c). e,f: Otx3 is not detectable in the midbrain and posterior forebrain of ROSA26eGFP-DTA/+;Wnt1-Cre compound embryos at 8.5 dpc (f). g,h: Double in situ hybridisation with Bf1 (arrowhead) and Hoxb1 (asterisk). Expression of these markers is detectable in ROSA26eGFP-DTA/+;Wnt1-Cre compound embryos (h), although quantity of brain tissue in between is severely reduced. i,j: Fgf8 expression appears normal in the anterior neural ridge of the ROSA26eGFP-DTA/+;Wnt1-Cre compound embryo (arrow), but there is no MHB expression of Fgf8 (black arrowheads). Fgf8 expression in the tail bud (white arrowheads) and the branchial region (white arrows) is normal in compound heterozygous embryos. Scale bars = 200 μm.

The extent of the ablation was analysed further by in situ hybridisation with Otx3, a neural marker expressed in midbrain and posterior forebrain territories with a sharp posterior limit at the MHB (Gogoi et al., 2002; Ohtoshi et al., 2002). No Otx3 expression was detected in ROSA26eGFP-DTA/+;Wnt1-Cre compound embryos, suggesting ablation of these regions at 8.5 dpc (Fig. 4e,f). The presence of forebrain and hindbrain tissue was demonstrated by analysis of Bf1, Fgf8, and Hoxb1 expression (Tao and Lai, 1992; Crossley and Martin, 1995; Gavalas et al., 1998). Both forebrain markers, Bf1 and Fgf8, were expressed in the developing telencephalon in ROSA26eGFP-DTA/+;Wnt1-Cre embryos in a similar pattern to that observed in control littermates (Fig. 4g-j). However, Fgf8 expression in the MHB was not detectable, suggesting a requirement of Wnt1 expressing cells for normal Fgf8 expression (Matsunaga et al., 2002). Hoxb1 expression in rhombomere 4 was indistinguishable between mutants and control littermates (Fig. 4g,h).

We estimate a time lapse of less than 24 h between the onset of Cre expression and cell death in both ROSA26eGFP-DTA/+;Nkx2.5Cre/+ and ROSA26eGFP-DTA/+;Wnt1-Cre embryos. Strong lacZ expression is first detected in ROSA26-lacZ;Nkx2.5Cre/+ embryos only at the 1-2 somite stage (Moses et al., 2001). Since we first observed the absence of the heart in ROSA26eGFP-DTA/+;Nkx2.5Cre/+ at the 8-10 somite stage (data not shown), this suggests a minimum time lapse of around 14-18 h to achieve heart ablation.

The detection of Wnt1 and Cre expression in ROSA26eGFP-DTA/+;Wnt1-Cre embryos (Fig. 4a-d) can be explained by taking into account the dynamic pattern of Wnt1 expression during early development. Wnt1 is first expressed in the prospective midbrain region of the presomitic embryo (Echelard et al., 1994; Rowitch et al., 1998). By the 5-6 somite stage embryo, Wnt1 transcripts are still detected in the prospective midbrain, but have now also appeared in the dorsal aspects of the hindbrain. A few hours later (8-10 somites) Wnt1 expression is, in addition, observable in the spinal cord (Echelard et al., 1994; Rowitch et al., 1998). Therefore, there is an anterior to posterior wave of Wnt1 expression during these early stages of development. The Wnt1-Cre construct used to make the Wnt1-Cre mouse line has been shown to drive Cre expression in the same manner as the endogenous Wnt1 (Jiang et al., 2000). Therefore, the prediction is that in ROSA26eGFP-DTA/+;Wnt1-Cre compound embryos, DTA expression, and therefore cell ablation, should occur first in the midbrain region, later in the dorsal hindbrain, and finally in the dorsal spinal cord. This fits well with our observations (Fig. 4a-d and data not shown). We estimate a minimum time lapse of around 16-20 h between the onset of Cre expression in the midbrain (presomitic stages) and cell ablation (8-10 somites), which is similar to that observed in ROSA26eGFP-DTA/+;Nkx2.5Cre/+ mutants.

Similar DTA mouse lines to the one described here have been generated recently (Brockschnieder et al., 2004; Matsumura et al., 2004; Sato and Tanigawa, 2005). In comparison with the mouse line of Brockschnieder et al. (2004), our mouse line offers the advantage of expressing eGFP rather than lacZ ubiquitously. Moreover, Cre-mediated recombination excises not only eGFP and the polyadenylation signals, but also the Neo cassette in our construct. In contrast, in the DTA mouse of Brockschnieder et al. the excision leaves behind the entire Neo cassette, which might impair normal transcription of the ROSA26 locus, reducing expression of DTA. This could explain the longer time lapse between the onset of Cre expression and cell death in their system (36-48 h).

The DTA mice of Matsumura et al. (2004) and Sato and Tanigawa (2005) are transgenic lines that were generated by microinjection into the pronuclei of fertilised eggs. As pronucleus microinjection normally leads to tandem incorporation of the transgene, Cre recombination can cause aberrant expression of DTA, depending on which loxP sites are utilised. This might explain the incomplete ablation of Cre-expressing cells in one of the DTA mouse lines. In contrast, the mouse described here was obtained by homologous recombination in ES cells, which enables specific recombination between the two unique loxP sites and, so, expression of DTA in all Cre-expressing cells. The time between initial Cre expression and cell death was not estimated in these two transgenic DTA mouse lines (Matsumura et al., 2004; Sato and Tanigawa, 2005).

In conclusion, we have characterised a ROSA26-eGFP-DTA mouse line that can be used as a general GFP reporter and as a tool for genetic cell ablation experiments. We have proven that this system can be used successfully to ablate specific embryonic regions. We expect this system to be useful not only in developmental biology studies, but also in the field of stem cell research and human medicine, for example, to generate cell-depleted animals and model degenerative diseases caused by increased apoptosis in mice.

MATERIALS AND METHODS

Generation of the Targeting Construct and the ROSA-eGFP-DTA Mouse Line

The components of the ROSA26 targeting vectors were a gift from P. Soriano and S. Srinivas (Soriano, 1999; Srinivas et al., 2001). To avoid possible interference with translation of DTA due to the presence of an ATG within the loxP sites, we generated a pBigT-invloxP by inverting the orientation of both loxP sites. Therefore, the first ATG in our construct after loxP recombination is the DTA initiation codon. We inserted eGFP (ClonTech, Palo Alto, CA) downstream of the splice acceptor and the 5′ loxP of the pBigT-invloxP, followed by a PGK Neo cassette, a triple SV40 polyadenylation signal, and DTA. DTA was obtained from I. Maxwell (Maxwell et al., 1986; Harrison et al., 1991), but we corrected its C-terminus by polymerase chain reaction (PCR) to generate the same sequence of DTA as described originally (Maxwell et al., 1987). The PacI-AscI DNA fragment of pBigT-invloxP was cloned into the ROSA26PA to generate the final targeting vector (Fig. 1a).

CCE ES cells (129/SvEv) were electroporated with the linearised construct depicted in Figure 1 and a total of 300 clones were picked, expanded, and frozen following standard protocols (Martinez-Barbera et al., 2000). Homologous recombination in the ROSA26 locus was identified by Southern blot using an external probe. This probe is a 140-bp DNA fragment that was excised from the pROSA26-5′ plasmid by EcoRI/HindIII digestion (Soriano, 1999; Srinivas et al., 2001). A total of six clones showed the correct band pattern with the external probe (Fig. 1b) and a Neo probe (data not shown). Three clones were used for blastocyst injection (C57BL6/J background) and all three gave germline transmission. ROSA26eGFP-DTA/+ heterozygous mice were intercrossed to generate ROSA26eGFP-DTA/eGFP-DTA homozygous mice. Mice of these genotypes were viable in both C57BL6/J and CD1 backgrounds.

Genotyping of Mice and Embryos

Genotyping was carried out by Southern blot and PCR as described (Soriano, 1999; Srinivas et al., 2001). Primers were R26R1 (5′-aaagtcgctctgagttgttat-3′), R26R2 (5′-gcgaagagtttgtcctcaacc-3′), and R26R3 (5′-ggagcgggagaaatggatatg-3′). Embryos were genotyped after in situ hybridisation as described (Martinez-Barbera et al., 2000). Detailed protocols are available upon request.

In Situ Hybridisation and Histology

In situ hybridisation was performed as described (Martinez-Barbera et al., 2000) using probes to α-cardiac actin, Cre, Wnt1, Otx3, Bf1, Nkx2.5, Hoxb1, and Fgf8. For histology, embryos were fixed in 4% paraformaldehyde, dehydrated in ethanol, embedded in wax, and sectioned at 8 μm as described (Martinez-Barbera et al., 2000).

ACKNOWLEDGMENTS

We thank P. Soriano, S. Shrinivas, and I. Maxwell for providing the reagents required for the generation of the targeting construct. Probes were obtained from G. Martin (Fgf8), A. Simeone (Cre and Hoxb1), A. Lumsden (Otx3), E. Lai (Bf1), A. McMahon (Wnt1), M. Buckingham (alpha-cardiac actin), and R. Harvey (Nkx2.5). We also thank A. McMahon and R. Schwartz for providing the Wnt1-Cre and Nkx2.5-Cre mouse lines, respectively. The authors thank A. Gavalas for help in the generation of the pBigT-invloxP and E. Sajedi, P. Riley, and N. Smart for discussions and assistance in some experiments.

Contract grant sponsor: Wellcome Trust.

Footnotes

The first two authors contributed equally to this work.

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