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Multiple phenotypes in adult mice following inactivation of the Coxsackievirus and Adenovirus Receptor (Car) gene.

Pazirandeh A, Sultana T, Mirza M, Rozell B, Hultenby K, Wallis K, Vennström B, Davis B, Arner A, Heuchel R, Löhr M, Philipson L, Sollerbrant K - PLoS ONE (2011)

Bottom Line: To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues.All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia.These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institutet for Cancer Research, Stockholm Branch, Stockholm, Sweden.

ABSTRACT
To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhibited a complete atrio-ventricular block and abnormal thymopoiesis. These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo.

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Construction of a conditional Car knockout mouse.(A) Schematic presentation of the targeting strategy. Tamoxifen is required to produce a Car  allele (cKO). (B) PCR analysis of genomic DNA isolated from the mouse tail. The genotype of wt (+/+), heterozygote (F/+) and homozygote (F/F) mice is indicated. The 261 bp and 211 bp PCR products corresponds to floxed and wt alleles respectively. (C) Western blotting of protein extracts prepared from pancreas shows that insertion of loxP sites and the Cre transgene do not affect CAR protein levels in the absence of tamoxifen. Genotypes and molecular weight markers are indicated. The CAR-specific antibody RP291 was used to detect CAR. The same result was obtained with the antibody RP1284 (data not shown). Calnexin was used as a loading control. The experiment was performed >3 times. (D) Indirect immunofluorescence on sections from large intestine was used to demonstrate that insertion of loxP sites did not affect the subcellular localization of the CAR protein in the absence of tamoxifen. Genotypes are indicated. The CAR-specific antibody RP291 was used to detect CAR (green). The same result was obtained when the IG1 antibody was used to detect CAR (data not shown). DAPI was used to visualize nuclei (blue). Magnification was 40×. (i) and (ii) are enlargements of the boxed areas in the +/+ and F/F image, respectively. The experiment was performed 3 times.
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pone-0020203-g001: Construction of a conditional Car knockout mouse.(A) Schematic presentation of the targeting strategy. Tamoxifen is required to produce a Car allele (cKO). (B) PCR analysis of genomic DNA isolated from the mouse tail. The genotype of wt (+/+), heterozygote (F/+) and homozygote (F/F) mice is indicated. The 261 bp and 211 bp PCR products corresponds to floxed and wt alleles respectively. (C) Western blotting of protein extracts prepared from pancreas shows that insertion of loxP sites and the Cre transgene do not affect CAR protein levels in the absence of tamoxifen. Genotypes and molecular weight markers are indicated. The CAR-specific antibody RP291 was used to detect CAR. The same result was obtained with the antibody RP1284 (data not shown). Calnexin was used as a loading control. The experiment was performed >3 times. (D) Indirect immunofluorescence on sections from large intestine was used to demonstrate that insertion of loxP sites did not affect the subcellular localization of the CAR protein in the absence of tamoxifen. Genotypes are indicated. The CAR-specific antibody RP291 was used to detect CAR (green). The same result was obtained when the IG1 antibody was used to detect CAR (data not shown). DAPI was used to visualize nuclei (blue). Magnification was 40×. (i) and (ii) are enlargements of the boxed areas in the +/+ and F/F image, respectively. The experiment was performed 3 times.

Mentions: We constructed a floxed Car allele that can be disrupted at any chosen time point by tamoxifen-regulated Cre-mediated recombination in vivo (Fig. 1A). Genotypes were determined by PCR amplification of genomic DNA isolated from the tail (Fig. 1B and data not shown).


Multiple phenotypes in adult mice following inactivation of the Coxsackievirus and Adenovirus Receptor (Car) gene.

Pazirandeh A, Sultana T, Mirza M, Rozell B, Hultenby K, Wallis K, Vennström B, Davis B, Arner A, Heuchel R, Löhr M, Philipson L, Sollerbrant K - PLoS ONE (2011)

Construction of a conditional Car knockout mouse.(A) Schematic presentation of the targeting strategy. Tamoxifen is required to produce a Car  allele (cKO). (B) PCR analysis of genomic DNA isolated from the mouse tail. The genotype of wt (+/+), heterozygote (F/+) and homozygote (F/F) mice is indicated. The 261 bp and 211 bp PCR products corresponds to floxed and wt alleles respectively. (C) Western blotting of protein extracts prepared from pancreas shows that insertion of loxP sites and the Cre transgene do not affect CAR protein levels in the absence of tamoxifen. Genotypes and molecular weight markers are indicated. The CAR-specific antibody RP291 was used to detect CAR. The same result was obtained with the antibody RP1284 (data not shown). Calnexin was used as a loading control. The experiment was performed >3 times. (D) Indirect immunofluorescence on sections from large intestine was used to demonstrate that insertion of loxP sites did not affect the subcellular localization of the CAR protein in the absence of tamoxifen. Genotypes are indicated. The CAR-specific antibody RP291 was used to detect CAR (green). The same result was obtained when the IG1 antibody was used to detect CAR (data not shown). DAPI was used to visualize nuclei (blue). Magnification was 40×. (i) and (ii) are enlargements of the boxed areas in the +/+ and F/F image, respectively. The experiment was performed 3 times.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108585&req=5

pone-0020203-g001: Construction of a conditional Car knockout mouse.(A) Schematic presentation of the targeting strategy. Tamoxifen is required to produce a Car allele (cKO). (B) PCR analysis of genomic DNA isolated from the mouse tail. The genotype of wt (+/+), heterozygote (F/+) and homozygote (F/F) mice is indicated. The 261 bp and 211 bp PCR products corresponds to floxed and wt alleles respectively. (C) Western blotting of protein extracts prepared from pancreas shows that insertion of loxP sites and the Cre transgene do not affect CAR protein levels in the absence of tamoxifen. Genotypes and molecular weight markers are indicated. The CAR-specific antibody RP291 was used to detect CAR. The same result was obtained with the antibody RP1284 (data not shown). Calnexin was used as a loading control. The experiment was performed >3 times. (D) Indirect immunofluorescence on sections from large intestine was used to demonstrate that insertion of loxP sites did not affect the subcellular localization of the CAR protein in the absence of tamoxifen. Genotypes are indicated. The CAR-specific antibody RP291 was used to detect CAR (green). The same result was obtained when the IG1 antibody was used to detect CAR (data not shown). DAPI was used to visualize nuclei (blue). Magnification was 40×. (i) and (ii) are enlargements of the boxed areas in the +/+ and F/F image, respectively. The experiment was performed 3 times.
Mentions: We constructed a floxed Car allele that can be disrupted at any chosen time point by tamoxifen-regulated Cre-mediated recombination in vivo (Fig. 1A). Genotypes were determined by PCR amplification of genomic DNA isolated from the tail (Fig. 1B and data not shown).

Bottom Line: To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues.All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia.These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institutet for Cancer Research, Stockholm Branch, Stockholm, Sweden.

ABSTRACT
To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhibited a complete atrio-ventricular block and abnormal thymopoiesis. These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo.

Show MeSH
Related in: MedlinePlus