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Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan.

Muro AF, Chauhan AK, Gajovic S, Iaconcig A, Porro F, Stanta G, Baralle FE - J. Cell Biol. (2003)

Bottom Line: However, the precise role of the FN isoforms is poorly understood.One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging.Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon.

View Article: PubMed Central - PubMed

Affiliation: International Center for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy.

ABSTRACT
Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

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Strategy for the generation of mouse strains lacking regulated splicing at the EDA exon of the FN gene. (A) Partial maps of the wild-type FN allele, the targeting vector, the targeted allele, the EDA-floxed allele, and the EDA- alleles. Exons, Neo-TK, and DTA are shown as gray, white, and dotted boxes, respectively. The EDA exon and the “loxP” sites are indicated as a black square and as white triangles, respectively. N and H indicate NcoI and HindIII sites, respectively. The recombinant ES cells were transiently transfected with a CRE-recombinase expressing plasmid, and EDA+/wt heterozygote cells were used for blastocyst microinjection. EDA+/wt mice were mated with a CRE-expressing transgenic mouse strain to obtain the EDA−/wt mice. The asterisks indicate the NdeI sites. The 4.0-, 2.7-, and 2.0-kb DNA fragments (EDAwt, EDA+, and EDA− alleles, respectively) obtained after digestion with HindIII and Southern blot hybridization with the internal probe are indicated. (B) Southern blot screening of the different mouse genotypes. EDAwt, EDA+, and EDA− bands are indicated. (C) RT-PCR analysis of total RNA prepared from EDAwt/wt, EDA+/+, and EDA−/− from liver, brain, and kidney is shown. EDA+ and EDA− bands are indicated (805 and 535 bp, respectively).
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fig1: Strategy for the generation of mouse strains lacking regulated splicing at the EDA exon of the FN gene. (A) Partial maps of the wild-type FN allele, the targeting vector, the targeted allele, the EDA-floxed allele, and the EDA- alleles. Exons, Neo-TK, and DTA are shown as gray, white, and dotted boxes, respectively. The EDA exon and the “loxP” sites are indicated as a black square and as white triangles, respectively. N and H indicate NcoI and HindIII sites, respectively. The recombinant ES cells were transiently transfected with a CRE-recombinase expressing plasmid, and EDA+/wt heterozygote cells were used for blastocyst microinjection. EDA+/wt mice were mated with a CRE-expressing transgenic mouse strain to obtain the EDA−/wt mice. The asterisks indicate the NdeI sites. The 4.0-, 2.7-, and 2.0-kb DNA fragments (EDAwt, EDA+, and EDA− alleles, respectively) obtained after digestion with HindIII and Southern blot hybridization with the internal probe are indicated. (B) Southern blot screening of the different mouse genotypes. EDAwt, EDA+, and EDA− bands are indicated. (C) RT-PCR analysis of total RNA prepared from EDAwt/wt, EDA+/+, and EDA−/− from liver, brain, and kidney is shown. EDA+ and EDA− bands are indicated (805 and 535 bp, respectively).

Mentions: To generate an FN allele devoid of alternative splicing in the EDA exon, the wild-type EDA exon in the FN gene was replaced with a “floxed” EDA exon having both the 5′ and 3′ optimized splice sites described in Fig. S1 (available at http:www.jcb.org/cgi/content/full/jcb.200212079/DC1). After electroporation of 129 Sv/J ES cells with the targeting construct (Fig. 1Figure 1.


Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan.

Muro AF, Chauhan AK, Gajovic S, Iaconcig A, Porro F, Stanta G, Baralle FE - J. Cell Biol. (2003)

Strategy for the generation of mouse strains lacking regulated splicing at the EDA exon of the FN gene. (A) Partial maps of the wild-type FN allele, the targeting vector, the targeted allele, the EDA-floxed allele, and the EDA- alleles. Exons, Neo-TK, and DTA are shown as gray, white, and dotted boxes, respectively. The EDA exon and the “loxP” sites are indicated as a black square and as white triangles, respectively. N and H indicate NcoI and HindIII sites, respectively. The recombinant ES cells were transiently transfected with a CRE-recombinase expressing plasmid, and EDA+/wt heterozygote cells were used for blastocyst microinjection. EDA+/wt mice were mated with a CRE-expressing transgenic mouse strain to obtain the EDA−/wt mice. The asterisks indicate the NdeI sites. The 4.0-, 2.7-, and 2.0-kb DNA fragments (EDAwt, EDA+, and EDA− alleles, respectively) obtained after digestion with HindIII and Southern blot hybridization with the internal probe are indicated. (B) Southern blot screening of the different mouse genotypes. EDAwt, EDA+, and EDA− bands are indicated. (C) RT-PCR analysis of total RNA prepared from EDAwt/wt, EDA+/+, and EDA−/− from liver, brain, and kidney is shown. EDA+ and EDA− bands are indicated (805 and 535 bp, respectively).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172721&req=5

fig1: Strategy for the generation of mouse strains lacking regulated splicing at the EDA exon of the FN gene. (A) Partial maps of the wild-type FN allele, the targeting vector, the targeted allele, the EDA-floxed allele, and the EDA- alleles. Exons, Neo-TK, and DTA are shown as gray, white, and dotted boxes, respectively. The EDA exon and the “loxP” sites are indicated as a black square and as white triangles, respectively. N and H indicate NcoI and HindIII sites, respectively. The recombinant ES cells were transiently transfected with a CRE-recombinase expressing plasmid, and EDA+/wt heterozygote cells were used for blastocyst microinjection. EDA+/wt mice were mated with a CRE-expressing transgenic mouse strain to obtain the EDA−/wt mice. The asterisks indicate the NdeI sites. The 4.0-, 2.7-, and 2.0-kb DNA fragments (EDAwt, EDA+, and EDA− alleles, respectively) obtained after digestion with HindIII and Southern blot hybridization with the internal probe are indicated. (B) Southern blot screening of the different mouse genotypes. EDAwt, EDA+, and EDA− bands are indicated. (C) RT-PCR analysis of total RNA prepared from EDAwt/wt, EDA+/+, and EDA−/− from liver, brain, and kidney is shown. EDA+ and EDA− bands are indicated (805 and 535 bp, respectively).
Mentions: To generate an FN allele devoid of alternative splicing in the EDA exon, the wild-type EDA exon in the FN gene was replaced with a “floxed” EDA exon having both the 5′ and 3′ optimized splice sites described in Fig. S1 (available at http:www.jcb.org/cgi/content/full/jcb.200212079/DC1). After electroporation of 129 Sv/J ES cells with the targeting construct (Fig. 1Figure 1.

Bottom Line: However, the precise role of the FN isoforms is poorly understood.One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging.Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon.

View Article: PubMed Central - PubMed

Affiliation: International Center for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy.

ABSTRACT
Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

Show MeSH
Related in: MedlinePlus