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Perlecan maintains the integrity of cartilage and some basement membranes.

Costell M, Gustafsson E, Aszódi A, Mörgelin M, Bloch W, Hunziker E, Addicks K, Timpl R, Fässler R - J. Cell Biol. (1999)

Bottom Line: As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function.The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly.Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Pathology, Lund University, S-221 85 Lund, Sweden.

ABSTRACT
Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

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Targeting strategy, Southern blots, PCR, and RIA analysis of ES cells and mice lacking perlecan. (A) Structure of the wild-type perlecan allele, targeting construct, and targeted perlecan allele before and after cre-loxP–mediated deletion. The dark boxes show exons of the perlecan gene, the open box shows the neo-tk cassette, and the loxP sites are indicated as triangles. Probe 1 was used to detect homologous recombination, and probe 2 was used to identify the  allele. Restriction sites are as follows: E, EcoRI; B, BamHI; and H, HindIII. (B) Southern blot analysis of EcoRI-digested genomic DNA derived from wild-type, heterozygous, and homozygous mutant mice hybridized with probe 1. (C) Detection of mutant mRNA lacking exon 6 by RT-PCR using the total RNA prepared from wild-type, heterozygous, and homozygous ES cells. Primers from exons 5 and 8 amplified 640 bp from the wild-type and 479 bp from the mutant allele. PCR products were probed with exon 7–specific radiolabeled oligonucleotide. (D) Radioimmunoassay measurement of laminin-1, nidogen-1, and perlecan in the serum-free medium derived from wild-type and mutant ES cells. Values are means ± SD of triplicate culture dishes and are expressed as micrograms per milliliter of supernatant.
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Figure 1: Targeting strategy, Southern blots, PCR, and RIA analysis of ES cells and mice lacking perlecan. (A) Structure of the wild-type perlecan allele, targeting construct, and targeted perlecan allele before and after cre-loxP–mediated deletion. The dark boxes show exons of the perlecan gene, the open box shows the neo-tk cassette, and the loxP sites are indicated as triangles. Probe 1 was used to detect homologous recombination, and probe 2 was used to identify the allele. Restriction sites are as follows: E, EcoRI; B, BamHI; and H, HindIII. (B) Southern blot analysis of EcoRI-digested genomic DNA derived from wild-type, heterozygous, and homozygous mutant mice hybridized with probe 1. (C) Detection of mutant mRNA lacking exon 6 by RT-PCR using the total RNA prepared from wild-type, heterozygous, and homozygous ES cells. Primers from exons 5 and 8 amplified 640 bp from the wild-type and 479 bp from the mutant allele. PCR products were probed with exon 7–specific radiolabeled oligonucleotide. (D) Radioimmunoassay measurement of laminin-1, nidogen-1, and perlecan in the serum-free medium derived from wild-type and mutant ES cells. Values are means ± SD of triplicate culture dishes and are expressed as micrograms per milliliter of supernatant.

Mentions: A 700-bp DNA fragment from the 5′ region of the mouse perlecan cDNA was used to screen a genomic library derived from a mouse D3/129 embryonic stem (ES) cell line (a gift from J.S. Mudgett, Merck Sharp & Dohme, NJ) to isolate perlecan genomic clones. The targeting construct (see Fig. 1 A) consisted of an 8-kb fragment containing exon 5, an expression cassette flanked by loxP sites in which the phosphoglycerate kinase promoter controls the expression of the neomycin (neo) gene and the Herpes simplex virus thymidine kinase (HSV-tk) gene, respectively, an 0.8-kb fragment containing exon 6 followed by a single loxP site and a 1.5-kb fragment containing exon 7 (for more detailed information contact: reinhard. fassler@pat.lu.se).


Perlecan maintains the integrity of cartilage and some basement membranes.

Costell M, Gustafsson E, Aszódi A, Mörgelin M, Bloch W, Hunziker E, Addicks K, Timpl R, Fässler R - J. Cell Biol. (1999)

Targeting strategy, Southern blots, PCR, and RIA analysis of ES cells and mice lacking perlecan. (A) Structure of the wild-type perlecan allele, targeting construct, and targeted perlecan allele before and after cre-loxP–mediated deletion. The dark boxes show exons of the perlecan gene, the open box shows the neo-tk cassette, and the loxP sites are indicated as triangles. Probe 1 was used to detect homologous recombination, and probe 2 was used to identify the  allele. Restriction sites are as follows: E, EcoRI; B, BamHI; and H, HindIII. (B) Southern blot analysis of EcoRI-digested genomic DNA derived from wild-type, heterozygous, and homozygous mutant mice hybridized with probe 1. (C) Detection of mutant mRNA lacking exon 6 by RT-PCR using the total RNA prepared from wild-type, heterozygous, and homozygous ES cells. Primers from exons 5 and 8 amplified 640 bp from the wild-type and 479 bp from the mutant allele. PCR products were probed with exon 7–specific radiolabeled oligonucleotide. (D) Radioimmunoassay measurement of laminin-1, nidogen-1, and perlecan in the serum-free medium derived from wild-type and mutant ES cells. Values are means ± SD of triplicate culture dishes and are expressed as micrograms per milliliter of supernatant.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169352&req=5

Figure 1: Targeting strategy, Southern blots, PCR, and RIA analysis of ES cells and mice lacking perlecan. (A) Structure of the wild-type perlecan allele, targeting construct, and targeted perlecan allele before and after cre-loxP–mediated deletion. The dark boxes show exons of the perlecan gene, the open box shows the neo-tk cassette, and the loxP sites are indicated as triangles. Probe 1 was used to detect homologous recombination, and probe 2 was used to identify the allele. Restriction sites are as follows: E, EcoRI; B, BamHI; and H, HindIII. (B) Southern blot analysis of EcoRI-digested genomic DNA derived from wild-type, heterozygous, and homozygous mutant mice hybridized with probe 1. (C) Detection of mutant mRNA lacking exon 6 by RT-PCR using the total RNA prepared from wild-type, heterozygous, and homozygous ES cells. Primers from exons 5 and 8 amplified 640 bp from the wild-type and 479 bp from the mutant allele. PCR products were probed with exon 7–specific radiolabeled oligonucleotide. (D) Radioimmunoassay measurement of laminin-1, nidogen-1, and perlecan in the serum-free medium derived from wild-type and mutant ES cells. Values are means ± SD of triplicate culture dishes and are expressed as micrograms per milliliter of supernatant.
Mentions: A 700-bp DNA fragment from the 5′ region of the mouse perlecan cDNA was used to screen a genomic library derived from a mouse D3/129 embryonic stem (ES) cell line (a gift from J.S. Mudgett, Merck Sharp & Dohme, NJ) to isolate perlecan genomic clones. The targeting construct (see Fig. 1 A) consisted of an 8-kb fragment containing exon 5, an expression cassette flanked by loxP sites in which the phosphoglycerate kinase promoter controls the expression of the neomycin (neo) gene and the Herpes simplex virus thymidine kinase (HSV-tk) gene, respectively, an 0.8-kb fragment containing exon 6 followed by a single loxP site and a 1.5-kb fragment containing exon 7 (for more detailed information contact: reinhard. fassler@pat.lu.se).

Bottom Line: As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function.The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly.Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Pathology, Lund University, S-221 85 Lund, Sweden.

ABSTRACT
Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

Show MeSH
Related in: MedlinePlus