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First published online 31 January 2006
doi: 10.1242/jcs.02800

Journal of Cell Science 119, 693-701 (2006)
Published by The Company of Biologists 2006
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Connexin31 cannot functionally replace connexin43 during cardiac morphogenesis in mice

Qingyi Zheng-Fischhöfer1, Alexander Ghanem2, Jung-Sun Kim3, Mark Kibschull4, Gaby Schwarz1, Jörg O. Schwab2, James Nagy5, Elke Winterhager4, Klaus Tiemann2 and Klaus Willecke1,*

1 Institut für Genetik, Universität Bonn, 53117 Bonn, Germany
2 Medizinische Klinik und Poliklinik II, Universitätsklinikum Bonn, 53105 Bonn, Germany
3 University of Ulsan, College of Medicine, Seoul, 138-736 Republic of Korea
4 Institut für Anatomie, Universität Duisburg-Essen, 45122 Essen, Germany
5 Department of Physiology, University of Manitoba, Canada


Figure 1
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  		Fig. 1. Generation and characterization of Cx4331/31 mice. (A) Vector pCx43KI31 DNA was transfected into HM-1 embryonic stem cells. Homologous recombination was tested by PCR and Southern blot analysis. Correctly recombined clones were injected into C57BL/6 blastocysts to generate first chimeras and in the next generation mice carrying the floxCx43KI31neo allele. By means of Cre activity, the coding region of connexin43 and the frt-flanked selection cassette were deleted, resulting in the Cx43KI31 allele. Thus, in mice carrying this allele, Cx31 is expressed under control of Cx43 regulatory elements. (B) Southern blot analysis of PstI digested Cx43+/+, Cx43+/31 and Cx4331/31 DNA using the external Cx43 probe. A 8 kb fragment is indicative of the Cx43 wild-type allele, whereas a 2.9 kb fragment indicates the Cx43KI31 allele. (C) Southern blot analysis of PstI digested Cx43+/+, Cx43+/31 and Cx4331/31 DNA using the internal Cx43 probe. Both Cx43+/+ and Cx43+/31 DNA showed a wild-type fragment of 8 kb, but Cx4331/31 did not. (D) Southern blot analysis of HindIII digested Cx43+/+, Cx43+/31 and Cx4331/31 DNA using the internal Cx31 probe. All three genotypes yielded a 12 kb wild-type fragment, whereas Cx43+/31 and Cx4331/31 showed an additional 5.3 kb recombinant fragment. (E) PCR analysis of different genotypes. Lane 1: 100 bp marker; lane 2: a 2.1 kb amplicon indicates that homologous recombination has occurred in the floxCx43KI31neo allele using primer 1 (Cx31Ki2) and 2 (Cx43-3'-RO3); lane 3: using primer 3 (Cx43/31fw) and primer 4 (Cx43/31rev), the wild-type allele yielded a 326 bp DNA fragment, whereas floxCx43KI31neo allele gave rise to a 375 bp fragment due to an additional loxP site; lane 4: after Cre activity, a 736 bp amplicon was generated using primer 3 (Cx43/31fw) and primer 5 (Cx31Ki3rev); lane 5-7: a multiplex PCR was established using primer 6 (Cx43-3'-HO2), primer 7 (Cx31Ki3) and primer 8 (Cx43-3'-RO4). A 381 bp fragment indicates the wild-type allele and a 615 bp fragment the knock-in allele. Lane 6: the Cx43+/31 genotype.

 

Figure 2
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  		Fig. 2. Western blot analyses of Cx43 and Cx31 in heart and brain lysates. Lane 1: Cx43+/+; lane 2: Cx43+/31; lane 3: Cx4331/31. Cx43 protein was not detected in Cx4331/31 animals (lane 3). Cx31 was not found in the Cx43+/+ heart and brain (lane 1), whereas it was expressed in Cx43+/31 mice and there was nearly double the amount in Cx4331/31 animals (lane 2 and lane 3). Loading of equal amounts of homogenates was verified by re-immunostaining with monoclonal anti-tubulin.

 

Figure 3
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  		Fig. 3. Immunofluorescence analysis of Cx43 and Cx31 in heart. Cryosections of Cx43+/+ (A,D), Cx43+/31 (B,E) and Cx4331/31 (C,F) hearts at P0 were immunolabeled with anti-Cx43 (A-C, red), anti-Cx31 (D-F, green) and counterstained with propidium iodide (shown as blue nuclear staining). Cx43 was expressed in Cx43+/+ and Cx43+/31 ventricles, but not in Cx4331/31 ventricles. Cx31 was not detected in Cx43+/+ hearts, but in Cx43+/31 and Cx4331/31 ventricles. Bars, 20 µm.

 

Figure 4
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  		Fig. 4. Double immunofluorescence analysis using monoclonal anti-Cx43 and polyclonal anti-Cx31 antibodies. (A-C) Cx43+/31 P0 and (D-F) adult ventricles. Cx43 staining is shown in red (A,D), Cx31 staining in green (B,E). C is an overlay of A and B; F is an overlay of D and E. Bars, 20 µm.

 

Figure 5
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  		Fig. 5. Cx4331/31 mice die postnatally. Litters of 10 heterozygous matings (86 pups) were used in the analysis. Cx4331/31 animals were born at the expected Mendelian ratio of 24.4% (21 animals), but died before P10.

 

Figure 6
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  		Fig. 6. Transverse sections through P4 hearts of Cx43+/+ (A), Cx43+/31 (B) and Cx4331/31 mice (C). The heart of the Cx4331/31 mouse showed irregular hypertrophic trabeculae with abnormal pouches (asterisks) in the subpulmonary outlet of the right ventricle. The heart of the Cx43+/31 mouse was almost normal. aa: ascending aorta; la, left atrium; ra, right atrium; rv, right ventricle; pv, pulmonary valve. Bar, 500 µm.

 

Figure 7
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  		Fig. 7. Representative signal-averaged ECG recordings performed at P0. Cx43+/+ (A) and Cx43+/31 mice (B) show normal ECG patterns. Note significant low voltage and prolonged P wave as well as QRS duration in Cx4331/31 mice (C).

 

Figure 8
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  		Fig. 8. Placental development in Cx4331/31 conceptuses. HE-stained paraffin sections of Cx43+/+ (A,C) and Cx4331/31 (B,D) placentas at E17.5. No differences in development and placental architecture were found. All trophoblast cell types [labyrinth (L); spongiotrophoblast (S); glycogen cells (G)] are normal in Cx4331/31 placenta compared with Cx43+/+ controls. Bars, 600 µm (A,B); 150 µm (C,D).

 

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© The Company of Biologists Ltd 2006