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Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome.

Cano-Gauci DF, Song HH, Yang H, McKerlie C, Choo B, Shi W, Pullano R, Piscione TD, Grisaru S, Soon S, Sedlackova L, Tanswell AK, Mak TW, Yeger H, Lockwood GA, Rosenblum ND, Filmus J - J. Cell Biol. (1999)

Bottom Line: These patients display pre- and postnatal overgrowth, and a varying range of dysmorphisms.Since BWS has been associated with biallelic expression of insulin-like growth factor II (IGF-II), it has been proposed that GPC3 is a negative regulator of IGF-II.In the particular case of the kidney, we demonstrate that there is an early and persistent developmental abnormality of the ureteric bud/collecting system due to increased proliferation of cells in this tissue element.

View Article: PubMed Central - PubMed

Affiliation: The Ontario Cancer Institute, Toronto, Ontario, M5G 2M9 Canada.

ABSTRACT
Glypicans are a family of heparan sulfate proteoglycans that are linked to the cell surface through a glycosyl-phosphatidylinositol anchor. One member of this family, glypican-3 (Gpc3), is mutated in patients with the Simpson-Golabi-Behmel syndrome (SGBS). These patients display pre- and postnatal overgrowth, and a varying range of dysmorphisms. The clinical features of SGBS are very similar to the more extensively studied Beckwith-Wiedemann syndrome (BWS). Since BWS has been associated with biallelic expression of insulin-like growth factor II (IGF-II), it has been proposed that GPC3 is a negative regulator of IGF-II. However, there is still no biochemical evidence indicating that GPC3 plays such a role.Here, we report that GPC3-deficient mice exhibit several of the clinical features observed in SGBS patients, including developmental overgrowth, perinatal death, cystic and dyplastic kidneys, and abnormal lung development. A proportion of the mutant mice also display mandibular hypoplasia and an imperforate vagina. In the particular case of the kidney, we demonstrate that there is an early and persistent developmental abnormality of the ureteric bud/collecting system due to increased proliferation of cells in this tissue element. The degree of developmental overgrowth of the GPC3-deficient mice is similar to that of mice deficient in IGF receptor type 2 (IGF2R), a well characterized negative regulator of IGF-II. Unlike the IGF2R-deficient mice, however, the levels of IGF-II in GPC3 knockouts are similar to those of the normal littermates.

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Gene targeting of the murine Gpc3 locus. (A) A 6.5-kb EcoRI fragment of the murine Gpc3 locus is shown (top). The SmaI fragment containing the promoter, the transcription start site (arrow), exon 1, and part of intron 1 is indicated. An EcoRI/Bg1II fragment containing the neo cassette, a neomycin gene with a PGK promoter, driving expression in the antisense direction, is shown in the middle. The mutated locus with the neo cassette replacing the SmaI fragment is shown below. Restriction enzyme sites are as follows: E, EcoRI; K, KpnI; S, SmaI. The probe (S1) hybridized to the 6.5-kb wild-type and 7.5-kb targeted allele fragments, as expected. PCR primer sites and orientation within the native and disrupted loci are indicated: a, PCRL3; b, PCR5; c, PCR2; d, PCRL2; and e, neo. (B) Southern blot analysis of EcoRI-digested genomic DNA extracted from tail biopsies of wild-type, heterozygous, and hemizygous mutant offspring (+/+, +/−, and −/, respectively). (C) Top, Northern blot analysis of GPC3 in wild-type (+/+) and (−/) E18.5 embryos. 10 μg total RNA was probed with a 2.2-kb full-length Gpc3 cDNA. Bottom, ethidium bromide staining demonstrates equal loading.
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Figure 1: Gene targeting of the murine Gpc3 locus. (A) A 6.5-kb EcoRI fragment of the murine Gpc3 locus is shown (top). The SmaI fragment containing the promoter, the transcription start site (arrow), exon 1, and part of intron 1 is indicated. An EcoRI/Bg1II fragment containing the neo cassette, a neomycin gene with a PGK promoter, driving expression in the antisense direction, is shown in the middle. The mutated locus with the neo cassette replacing the SmaI fragment is shown below. Restriction enzyme sites are as follows: E, EcoRI; K, KpnI; S, SmaI. The probe (S1) hybridized to the 6.5-kb wild-type and 7.5-kb targeted allele fragments, as expected. PCR primer sites and orientation within the native and disrupted loci are indicated: a, PCRL3; b, PCR5; c, PCR2; d, PCRL2; and e, neo. (B) Southern blot analysis of EcoRI-digested genomic DNA extracted from tail biopsies of wild-type, heterozygous, and hemizygous mutant offspring (+/+, +/−, and −/, respectively). (C) Top, Northern blot analysis of GPC3 in wild-type (+/+) and (−/) E18.5 embryos. 10 μg total RNA was probed with a 2.2-kb full-length Gpc3 cDNA. Bottom, ethidium bromide staining demonstrates equal loading.

Mentions: A 6.5-kb portion of the Gpc3 gene was cloned by screening a 129J-1 Dash II genomic library with the 5′ end of the Gpc3 cDNA. A SmaI fragment within the 6.5-kb genomic clone that included part of the promoter region and exon 1 was replaced by an EcoRI/BglII fragment containing the neomycin-resistance gene under the control of the PGK promoter, driving expression in the antisense direction (neo cassette). The final targeting vector carried the PGK–neo cassette flanked by part of the Gpc3 promoter and the first intron at the 5′ and 3′ ends, respectively (Fig. 1 A). The vector was linearized and used to electroporate 129/J embryonic stem (ES) cells. Transfected clones resistant to G418 were screened by PCR for homologous recombination. Two recombinant ES clones, 8D1 and 7F12, were then used for injections into C57BL/6 blastocysts. Germline transmission was identified by Southern blot analysis of EcoRI-digested genomic DNA using a SmaI/KpnI fragment (S1) of the genomic clone. Mice were subsequently genotyped using PCR as follows: the presence of the neo cassette was determined by amplifying a neo-specific band using primers PCR2 and neo, followed by primers PCRL2 and neo in nested PCR reactions; a single denaturation step (95°C for 5 min) was followed by 25 cycles (94°C for 2 min, 55°C for 2 min, and 72°C for 3 min), with a final single elongation step (72°C for 5 min). To detect the 420-bp wild-type fragment of the Gpc3 gene to distinguish heterozygous and homozygous mutants, primers PCRL3 and PCR5 were used in 25 cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 30 s), followed by a final elongation step (72°C for 5 min). Primers were: PCR2, 5′-GTGTGGTTCTATTGAATGGACCC-3′; neo, 5′-GCCAGCTCATTCCTCCACTCAT-3′; PCRL2, 5′-ACGTGACTATTTGTGGGTAGG-3′; PCRL2, 5′-ACGTGACTATTTGTGGGTAGG-3′; PCRL3, 5′-TTGCCACTCTCTCGTGCTCTCC-3′; and PCR5, 5′-CAGAGTCCATACTGTGCTTCC-3′.


Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome.

Cano-Gauci DF, Song HH, Yang H, McKerlie C, Choo B, Shi W, Pullano R, Piscione TD, Grisaru S, Soon S, Sedlackova L, Tanswell AK, Mak TW, Yeger H, Lockwood GA, Rosenblum ND, Filmus J - J. Cell Biol. (1999)

Gene targeting of the murine Gpc3 locus. (A) A 6.5-kb EcoRI fragment of the murine Gpc3 locus is shown (top). The SmaI fragment containing the promoter, the transcription start site (arrow), exon 1, and part of intron 1 is indicated. An EcoRI/Bg1II fragment containing the neo cassette, a neomycin gene with a PGK promoter, driving expression in the antisense direction, is shown in the middle. The mutated locus with the neo cassette replacing the SmaI fragment is shown below. Restriction enzyme sites are as follows: E, EcoRI; K, KpnI; S, SmaI. The probe (S1) hybridized to the 6.5-kb wild-type and 7.5-kb targeted allele fragments, as expected. PCR primer sites and orientation within the native and disrupted loci are indicated: a, PCRL3; b, PCR5; c, PCR2; d, PCRL2; and e, neo. (B) Southern blot analysis of EcoRI-digested genomic DNA extracted from tail biopsies of wild-type, heterozygous, and hemizygous mutant offspring (+/+, +/−, and −/, respectively). (C) Top, Northern blot analysis of GPC3 in wild-type (+/+) and (−/) E18.5 embryos. 10 μg total RNA was probed with a 2.2-kb full-length Gpc3 cDNA. Bottom, ethidium bromide staining demonstrates equal loading.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199732&req=5

Figure 1: Gene targeting of the murine Gpc3 locus. (A) A 6.5-kb EcoRI fragment of the murine Gpc3 locus is shown (top). The SmaI fragment containing the promoter, the transcription start site (arrow), exon 1, and part of intron 1 is indicated. An EcoRI/Bg1II fragment containing the neo cassette, a neomycin gene with a PGK promoter, driving expression in the antisense direction, is shown in the middle. The mutated locus with the neo cassette replacing the SmaI fragment is shown below. Restriction enzyme sites are as follows: E, EcoRI; K, KpnI; S, SmaI. The probe (S1) hybridized to the 6.5-kb wild-type and 7.5-kb targeted allele fragments, as expected. PCR primer sites and orientation within the native and disrupted loci are indicated: a, PCRL3; b, PCR5; c, PCR2; d, PCRL2; and e, neo. (B) Southern blot analysis of EcoRI-digested genomic DNA extracted from tail biopsies of wild-type, heterozygous, and hemizygous mutant offspring (+/+, +/−, and −/, respectively). (C) Top, Northern blot analysis of GPC3 in wild-type (+/+) and (−/) E18.5 embryos. 10 μg total RNA was probed with a 2.2-kb full-length Gpc3 cDNA. Bottom, ethidium bromide staining demonstrates equal loading.
Mentions: A 6.5-kb portion of the Gpc3 gene was cloned by screening a 129J-1 Dash II genomic library with the 5′ end of the Gpc3 cDNA. A SmaI fragment within the 6.5-kb genomic clone that included part of the promoter region and exon 1 was replaced by an EcoRI/BglII fragment containing the neomycin-resistance gene under the control of the PGK promoter, driving expression in the antisense direction (neo cassette). The final targeting vector carried the PGK–neo cassette flanked by part of the Gpc3 promoter and the first intron at the 5′ and 3′ ends, respectively (Fig. 1 A). The vector was linearized and used to electroporate 129/J embryonic stem (ES) cells. Transfected clones resistant to G418 were screened by PCR for homologous recombination. Two recombinant ES clones, 8D1 and 7F12, were then used for injections into C57BL/6 blastocysts. Germline transmission was identified by Southern blot analysis of EcoRI-digested genomic DNA using a SmaI/KpnI fragment (S1) of the genomic clone. Mice were subsequently genotyped using PCR as follows: the presence of the neo cassette was determined by amplifying a neo-specific band using primers PCR2 and neo, followed by primers PCRL2 and neo in nested PCR reactions; a single denaturation step (95°C for 5 min) was followed by 25 cycles (94°C for 2 min, 55°C for 2 min, and 72°C for 3 min), with a final single elongation step (72°C for 5 min). To detect the 420-bp wild-type fragment of the Gpc3 gene to distinguish heterozygous and homozygous mutants, primers PCRL3 and PCR5 were used in 25 cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 30 s), followed by a final elongation step (72°C for 5 min). Primers were: PCR2, 5′-GTGTGGTTCTATTGAATGGACCC-3′; neo, 5′-GCCAGCTCATTCCTCCACTCAT-3′; PCRL2, 5′-ACGTGACTATTTGTGGGTAGG-3′; PCRL2, 5′-ACGTGACTATTTGTGGGTAGG-3′; PCRL3, 5′-TTGCCACTCTCTCGTGCTCTCC-3′; and PCR5, 5′-CAGAGTCCATACTGTGCTTCC-3′.

Bottom Line: These patients display pre- and postnatal overgrowth, and a varying range of dysmorphisms.Since BWS has been associated with biallelic expression of insulin-like growth factor II (IGF-II), it has been proposed that GPC3 is a negative regulator of IGF-II.In the particular case of the kidney, we demonstrate that there is an early and persistent developmental abnormality of the ureteric bud/collecting system due to increased proliferation of cells in this tissue element.

View Article: PubMed Central - PubMed

Affiliation: The Ontario Cancer Institute, Toronto, Ontario, M5G 2M9 Canada.

ABSTRACT
Glypicans are a family of heparan sulfate proteoglycans that are linked to the cell surface through a glycosyl-phosphatidylinositol anchor. One member of this family, glypican-3 (Gpc3), is mutated in patients with the Simpson-Golabi-Behmel syndrome (SGBS). These patients display pre- and postnatal overgrowth, and a varying range of dysmorphisms. The clinical features of SGBS are very similar to the more extensively studied Beckwith-Wiedemann syndrome (BWS). Since BWS has been associated with biallelic expression of insulin-like growth factor II (IGF-II), it has been proposed that GPC3 is a negative regulator of IGF-II. However, there is still no biochemical evidence indicating that GPC3 plays such a role.Here, we report that GPC3-deficient mice exhibit several of the clinical features observed in SGBS patients, including developmental overgrowth, perinatal death, cystic and dyplastic kidneys, and abnormal lung development. A proportion of the mutant mice also display mandibular hypoplasia and an imperforate vagina. In the particular case of the kidney, we demonstrate that there is an early and persistent developmental abnormality of the ureteric bud/collecting system due to increased proliferation of cells in this tissue element. The degree of developmental overgrowth of the GPC3-deficient mice is similar to that of mice deficient in IGF receptor type 2 (IGF2R), a well characterized negative regulator of IGF-II. Unlike the IGF2R-deficient mice, however, the levels of IGF-II in GPC3 knockouts are similar to those of the normal littermates.

Show MeSH
Related in: MedlinePlus