Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development - PubMed (original) (raw)

Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development

M Tarutani et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Glycosylphosphatidylinositol (GPI)-anchored proteins are widely distributed on plasma membranes of eukaryotes. More than 50 GPI-anchored proteins have been shown to be spatiotemporally expressed in mice with a deficiency of GPI-anchor biosynthesis that causes embryonic lethality. Here, we examine the functional roles of GPI-anchored proteins in mouse skin using the Cre-loxP recombination system. We disrupted the Pig-a gene, an X-linked gene essential for GPI-anchor biosynthesis, in skin. The Cre-mediated Pig-a disruption occurred in skin at almost 100% efficiency in male mice bearing two identically orientated loxP sites within the Pig-a gene. Expression of GPI-anchored proteins was completely absent in the skin of these mice. The skin of such mutants looked wrinkled and more scaly than that of wild-type mice. Furthermore, histological examination of mutant mice showed that the epidermal horny layer was tightly packed and thickened. Electron microscopy showed that the intercellular space was narrow and there were many small vesicles embedded in the intercellular space that were not observed in equivalent wild-type mouse skin preparations. Mutant mice died within a few days after birth, suggesting that Pig-a function is essential for proper skin differentiation and maintenance.

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Figures

Figure 1

Figure 1

Targeted insertion of loxP sites into the Pig-a gene. (A) Part of the wild-type Pig-a locus showing the positions of exons 3–6, the targeting construct, and Pig-a allele containing the introduced loxP sites are shown. Solid and open boxes indicate the coding and noncoding exons, respectively. Restriction sites for _Bam_HI (B) and _Eco_RI (E) are indicated. (B) Cre-mediated disappearance of GPI-anchored proteins on the surface of ES cells containing the loxP sites in the Pig-a gene. The plasmid pMC-Cre (13) with hygr was transfected into clone 58. The surface expression of heat-stable antigens (HSAg) was examined before and after selection with 150 μg/ml of hygromycin B for 7 days. Five of the 12 clones lost surface GPI-anchored proteins after selection. A representative clone that lost GPI-anchored proteins is shown. (C) Southern hybridization of the DNA from the germ-line-transmitted and control mice. Genomic DNA was obtained from tails and digested with _Bam_HI. The 3.5- and 2.0-kb fragments represent targeted and endogenous alleles, respectively.

Figure 2

Figure 2

Creation of the K5Cre transgenic lines. (A) K5 promoter activity in cultured cell lines. The construct in which the luciferase gene was placed under the control of the K5 promoter (Upper) was transfected into a keratinocyte cell line (PAM 212) and a fibroblast line (NIH 3T3). The control vector in which the luciferase gene was driven by an simian virus 40 early promoter and its enhancer was also transfected into these cell lines. The luciferase activities were measured and are indicated as arbitrary units (Lower). (B) Structure of the K5Cre transgene. A 14-kb fragment of human K5 promoter used to control the expression of Cre transgene and the nuclear localizing signal was added at 5′ of Cre gene.

Figure 3

Figure 3

Tissue-specific disruption of the Pig-a gene and deficient expression of GPI-anchored proteins in Cre:_Pig-a_flox male mice. (A) Tissue-specific disruption of the Pig-a gene in Cre:_Pig-a_flox male mice. To estimate the efficiency of Pig-a disruption, allele-specific PCR was performed using the three primers shown on the right. Size markers are included. (B) Deficient expression of a GPI-anchored protein, heat-stable antigen (HSAg), in keratinocytes. Epidermis from 1-day-old mice was separated from dermis by trypsin, and then individual cells were obtained. The surface expression of HSAg was examined.

Figure 4

Figure 4

Macro- and microscopic changes of epidermis from wild-type and skin-specific, _Pig-a_-disrupted mouse (Cre:_Pig-a_flox male mouse). (A) Macroscopic appearance of the wild-type and the skin-specific, _Pig-a_-disrupted mouse. (B) Histological examination of the epidermis from the wild-type and the skin-specific, disrupted mouse. (Hematoxylin-eosin staining, ×200.) (C) Histological examination of the horny layers by electron microscopy. The vesicles in the horny layers are indicated by white arrows.

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