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Targeted deletion of the PEX2 peroxisome assembly gene in mice provides a model for Zellweger syndrome, a human neuronal migration disorder.

Faust PL, Hatten ME - J. Cell Biol. (1997)

Bottom Line: The mutant animals do not feed and are hypoactive and markedly hypotonic.They display abnormal peroxisomal biochemical parameters, including accumulations of very long chain fatty acids in plasma and deficient erythrocyte plasmalogens.Abnormal lipid storage is evident in the adrenal cortex, with characteristic lamellar-lipid inclusions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Developmental Neurobiology, The Rockefeller University, New York, New York 10021, USA. plf3@columbia.edu

ABSTRACT
Zellweger syndrome is a peroxisomal biogenesis disorder that results in abnormal neuronal migration in the central nervous system and severe neurologic dysfunction. The pathogenesis of the multiple severe anomalies associated with the disorders of peroxisome biogenesis remains unknown. To study the relationship between lack of peroxisomal function and organ dysfunction, the PEX2 peroxisome assembly gene (formerly peroxisome assembly factor-1) was disrupted by gene targeting. Homozygous PEX2-deficient mice survive in utero but die several hours after birth. The mutant animals do not feed and are hypoactive and markedly hypotonic. The PEX2-deficient mice lack normal peroxisomes but do assemble empty peroxisome membrane ghosts. They display abnormal peroxisomal biochemical parameters, including accumulations of very long chain fatty acids in plasma and deficient erythrocyte plasmalogens. Abnormal lipid storage is evident in the adrenal cortex, with characteristic lamellar-lipid inclusions. In the central nervous system of newborn mutant mice there is disordered lamination in the cerebral cortex and an increased cell density in the underlying white matter, indicating an abnormality of neuronal migration. These findings demonstrate that mice with a PEX2 gene deletion have a peroxisomal disorder and provide an important model to study the role of peroxisomal function in the pathogenesis of this human disease.

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Generation of PEX2-deficient mice. (A) Schematic  representation of the PEX2 genomic locus (top), targeting vector  (middle), and targeted allele (bottom). Exon sequences are indicated by the black boxes. Selectable neomycin resistance (NEO)  and thymidine kinase (TK) cassettes, under control of the PGK  promoter (shaded box), are shown. Dashed lines indicate the region of homology between locus and targeting vector. The origins  of the 5′ and 3′ probes (X and Y) and the size of the resulting restriction fragments (long arrows) used to identify the targeted ES  cell lines are shown. In the targeted locus, exon 5, containing the  translation initiation site and entire coding sequence, is replaced  by the NEO gene, resulting in a  allele. Bg, BglII; Bx, BstXI;  E, EcoRI; H, HindIII; X, XbaI. (B) Southern blots of genomic  DNA isolated from targeted ES cell lines. (Lanes 1–3) EcoRI digest gives a wild-type 6-kb fragment and targeted 9-kb fragment.  (Lanes 4–6) BglII digest gives a wild-type 13.8-kb fragment and  targeted 9.6-kb fragment. (C) PCR analysis of tail DNA of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice.  Primers used for the PCR (a–c) are indicated. M, BRL 1-kb ladder marker. (D) Northern blot hybridization analysis of RNA  from liver (lanes 1–3), brain (lanes 4–6), and kidney (lanes 7–9).  20 μg of total RNA from +/+ (lanes 1, 4, and 7), +/− (lanes 2, 5,  and 8), and −/− (lanes 3, 6, and 9) newborn mice were hybridized  with an exon 5 PEX2 genomic probe or GAPDH probe. Arrowheads indicate the three PEX2 transcripts identified. Size marker,  BRL RNA ladder.
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Figure 1: Generation of PEX2-deficient mice. (A) Schematic representation of the PEX2 genomic locus (top), targeting vector (middle), and targeted allele (bottom). Exon sequences are indicated by the black boxes. Selectable neomycin resistance (NEO) and thymidine kinase (TK) cassettes, under control of the PGK promoter (shaded box), are shown. Dashed lines indicate the region of homology between locus and targeting vector. The origins of the 5′ and 3′ probes (X and Y) and the size of the resulting restriction fragments (long arrows) used to identify the targeted ES cell lines are shown. In the targeted locus, exon 5, containing the translation initiation site and entire coding sequence, is replaced by the NEO gene, resulting in a allele. Bg, BglII; Bx, BstXI; E, EcoRI; H, HindIII; X, XbaI. (B) Southern blots of genomic DNA isolated from targeted ES cell lines. (Lanes 1–3) EcoRI digest gives a wild-type 6-kb fragment and targeted 9-kb fragment. (Lanes 4–6) BglII digest gives a wild-type 13.8-kb fragment and targeted 9.6-kb fragment. (C) PCR analysis of tail DNA of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. Primers used for the PCR (a–c) are indicated. M, BRL 1-kb ladder marker. (D) Northern blot hybridization analysis of RNA from liver (lanes 1–3), brain (lanes 4–6), and kidney (lanes 7–9). 20 μg of total RNA from +/+ (lanes 1, 4, and 7), +/− (lanes 2, 5, and 8), and −/− (lanes 3, 6, and 9) newborn mice were hybridized with an exon 5 PEX2 genomic probe or GAPDH probe. Arrowheads indicate the three PEX2 transcripts identified. Size marker, BRL RNA ladder.

Mentions: The PCR fragment was used to probe a 129SVJ mouse genomic λFIX II phage library (946309; Stratagene, La Jolla, CA) and a C57BL/6 P6 cerebellum cDNA library in λZAP (kindly provided by G. Dietz, Rockefeller University, New York, NY). These sequence data are available from Genbank/EMBL/DDBJ under accession number AF031128. The deduced genomic structure of the mouse PEX2 gene is shown in Fig. 1 A.


Targeted deletion of the PEX2 peroxisome assembly gene in mice provides a model for Zellweger syndrome, a human neuronal migration disorder.

Faust PL, Hatten ME - J. Cell Biol. (1997)

Generation of PEX2-deficient mice. (A) Schematic  representation of the PEX2 genomic locus (top), targeting vector  (middle), and targeted allele (bottom). Exon sequences are indicated by the black boxes. Selectable neomycin resistance (NEO)  and thymidine kinase (TK) cassettes, under control of the PGK  promoter (shaded box), are shown. Dashed lines indicate the region of homology between locus and targeting vector. The origins  of the 5′ and 3′ probes (X and Y) and the size of the resulting restriction fragments (long arrows) used to identify the targeted ES  cell lines are shown. In the targeted locus, exon 5, containing the  translation initiation site and entire coding sequence, is replaced  by the NEO gene, resulting in a  allele. Bg, BglII; Bx, BstXI;  E, EcoRI; H, HindIII; X, XbaI. (B) Southern blots of genomic  DNA isolated from targeted ES cell lines. (Lanes 1–3) EcoRI digest gives a wild-type 6-kb fragment and targeted 9-kb fragment.  (Lanes 4–6) BglII digest gives a wild-type 13.8-kb fragment and  targeted 9.6-kb fragment. (C) PCR analysis of tail DNA of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice.  Primers used for the PCR (a–c) are indicated. M, BRL 1-kb ladder marker. (D) Northern blot hybridization analysis of RNA  from liver (lanes 1–3), brain (lanes 4–6), and kidney (lanes 7–9).  20 μg of total RNA from +/+ (lanes 1, 4, and 7), +/− (lanes 2, 5,  and 8), and −/− (lanes 3, 6, and 9) newborn mice were hybridized  with an exon 5 PEX2 genomic probe or GAPDH probe. Arrowheads indicate the three PEX2 transcripts identified. Size marker,  BRL RNA ladder.
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Related In: Results  -  Collection

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Figure 1: Generation of PEX2-deficient mice. (A) Schematic representation of the PEX2 genomic locus (top), targeting vector (middle), and targeted allele (bottom). Exon sequences are indicated by the black boxes. Selectable neomycin resistance (NEO) and thymidine kinase (TK) cassettes, under control of the PGK promoter (shaded box), are shown. Dashed lines indicate the region of homology between locus and targeting vector. The origins of the 5′ and 3′ probes (X and Y) and the size of the resulting restriction fragments (long arrows) used to identify the targeted ES cell lines are shown. In the targeted locus, exon 5, containing the translation initiation site and entire coding sequence, is replaced by the NEO gene, resulting in a allele. Bg, BglII; Bx, BstXI; E, EcoRI; H, HindIII; X, XbaI. (B) Southern blots of genomic DNA isolated from targeted ES cell lines. (Lanes 1–3) EcoRI digest gives a wild-type 6-kb fragment and targeted 9-kb fragment. (Lanes 4–6) BglII digest gives a wild-type 13.8-kb fragment and targeted 9.6-kb fragment. (C) PCR analysis of tail DNA of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. Primers used for the PCR (a–c) are indicated. M, BRL 1-kb ladder marker. (D) Northern blot hybridization analysis of RNA from liver (lanes 1–3), brain (lanes 4–6), and kidney (lanes 7–9). 20 μg of total RNA from +/+ (lanes 1, 4, and 7), +/− (lanes 2, 5, and 8), and −/− (lanes 3, 6, and 9) newborn mice were hybridized with an exon 5 PEX2 genomic probe or GAPDH probe. Arrowheads indicate the three PEX2 transcripts identified. Size marker, BRL RNA ladder.
Mentions: The PCR fragment was used to probe a 129SVJ mouse genomic λFIX II phage library (946309; Stratagene, La Jolla, CA) and a C57BL/6 P6 cerebellum cDNA library in λZAP (kindly provided by G. Dietz, Rockefeller University, New York, NY). These sequence data are available from Genbank/EMBL/DDBJ under accession number AF031128. The deduced genomic structure of the mouse PEX2 gene is shown in Fig. 1 A.

Bottom Line: The mutant animals do not feed and are hypoactive and markedly hypotonic.They display abnormal peroxisomal biochemical parameters, including accumulations of very long chain fatty acids in plasma and deficient erythrocyte plasmalogens.Abnormal lipid storage is evident in the adrenal cortex, with characteristic lamellar-lipid inclusions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Developmental Neurobiology, The Rockefeller University, New York, New York 10021, USA. plf3@columbia.edu

ABSTRACT
Zellweger syndrome is a peroxisomal biogenesis disorder that results in abnormal neuronal migration in the central nervous system and severe neurologic dysfunction. The pathogenesis of the multiple severe anomalies associated with the disorders of peroxisome biogenesis remains unknown. To study the relationship between lack of peroxisomal function and organ dysfunction, the PEX2 peroxisome assembly gene (formerly peroxisome assembly factor-1) was disrupted by gene targeting. Homozygous PEX2-deficient mice survive in utero but die several hours after birth. The mutant animals do not feed and are hypoactive and markedly hypotonic. The PEX2-deficient mice lack normal peroxisomes but do assemble empty peroxisome membrane ghosts. They display abnormal peroxisomal biochemical parameters, including accumulations of very long chain fatty acids in plasma and deficient erythrocyte plasmalogens. Abnormal lipid storage is evident in the adrenal cortex, with characteristic lamellar-lipid inclusions. In the central nervous system of newborn mutant mice there is disordered lamination in the cerebral cortex and an increased cell density in the underlying white matter, indicating an abnormality of neuronal migration. These findings demonstrate that mice with a PEX2 gene deletion have a peroxisomal disorder and provide an important model to study the role of peroxisomal function in the pathogenesis of this human disease.

Show MeSH
Related in: MedlinePlus