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. 2005 Nov;43(3):129-35.
doi: 10.1002/gene.20162.

In vivo genetic ablation by Cre-mediated expression of diphtheria toxin fragment A

Anna Ivanova  1 , Massimo Signore, Nadia Caro, Nicholas D E Greene, Andrew J Copp, Juan Pedro Martinez-Barbera
Affiliations

In vivo genetic ablation by Cre-mediated expression of diphtheria toxin fragment A

Anna Ivanova et al. Genesis. 2005 Nov.

Abstract

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.

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Figures

FIG. 1
FIG. 1
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]
FIG. 2
FIG. 2
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
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]
FIG. 4
FIG. 4
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.
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