X Inactivation and Somatic Cell Selection Rescue Female Mice Carrying a Piga-Null Mutation

Proc. Natl. Acad. Sci. USA Vol. 96, pp. 7479–7483, June 1999 Medical Sciences X inactivation and somatic cell selection rescue female mice carrying a Piga-null mutation (Cre/loxPyglycosyl phosphatidylinositolyXp22yparoxysmal nocturnal hemoglobinuria) PETER KELLER*, GABI TREMML†,VITTORIO ROSTI†, AND MONICA BESSLER*‡ *Division of Hematology, Departments of Internal Medicine and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110; and †Department of Human Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 Edited by Ernest Beutler, The Scripps Research Institute, La Jolla, CA, and approved May 6, 1999 (received for review March 22, 1999) ABSTRACT A somatic mutation in the X linked PIGA some (‘‘Ohno’s law;’’ ref. 8), the murine Piga maps to X-F3/4. gene is responsible for the deficiency of glycosyl phosphati- Piga gene inactivation in murine embryonic stem (ES) cells dylinositol (GPI)-anchored proteins on blood cells from pa- followed by blastocyst injection is associated with a high rate tients with paroxysmal nocturnal hemoglobinuria. No inher- of early embryonic lethality and low chimerism in surviving ited form of GPI-anchor deficiency has been described. Be- animals (9, 10). Female mice heterozygous for a mutant Piga cause conventional Piga gene knockout is associated with high gene have never been obtained. To study the consequences of embryonic lethality in chimeric mice, we used the Cre/loxP a nonfunctional Piga gene and to address the issue of a system. We generated mice in which two loxP sites flank part maternally inherited Piga mutation, we generated mice carry- of Piga exon 2. After crossbreeding with female mice of the ing a Piga mutation using Cre/loxP-controlled DNA recombi- EIIa-cre strain, the floxed allele undergoes Cre-mediated nation (11). High efficiency of Piga gene recombination was recombination with high efficiency during early embryonic obtained by targeting Piga gene inactivation directly to the development. Because of X chromosome inactivation, female preimplantation female embryo. Because of X inactivation, offspring are mosaic for cells that express or lack GPI-linked newborn female mice are mosaic, with cells that express or lack proteins. Analysis of mosaic mice showed that in heart, lung, GPI-linked proteins. To assess the importance of PIGA in kidney, brain, and liver, mainly wild-type Piga is active, different organs, we determined the relative contribution of suggesting that these tissues require GPI-linked proteins. The cells expressing or lacking GPI-linked proteins. Female mice salient exceptions were spleen, thymus, and red blood cells, that had high efficiency of Piga gene recombination enabled us which had almost equal numbers of cells expressing the to further investigate the possibility of an inherited Piga gene wild-type or the recombined allele, implying that GPI-linked mutation. proteins are not essential for the derivation of these tissues. PIGA(2) cells had no growth advantage, suggesting that other MATERIALS AND METHODS factors are needed for their clonal dominance in patients with Production of the lox-Piga-lacZ Mice. The production of the paroxysmal nocturnal hemoglobinuria. lox-Piga-lacZ mice has been described in detail previously (12). In brief: two loxP sites were introduced by homologous re- The gene PIGA (phosphatidylinositol glycan class A) encodes combination into the Piga locus of 129SV-derived ES cells CJ7, a subunit of the a1–6-N-acetylglucosaminyltransferase com- flanking 662 bp of exon 2 and 1.6 kb of the adjoining intron 2. plex, an enzyme essential for the biosynthesis of glycosyl In addition, the coding region of the lacZ gene was inserted phosphatidylinositol (GPI) anchors (1, 2). In paroxysmal noc- into intron 2 in such a way that in the lox-Piga-lacZ configu- turnal hemoglobinuria (PNH), an acquired hemolytic anemia, ration (floxed Piga gene), LacZ is not expressed and does not a somatic mutation in the PIGA gene causes a proportion of interfere the function of PIGA. However, after Cre-mediated blood cells to be deficient in all GPI-linked molecules (3, 4). Piga recombination (lox-DPiga-lacZ), lacZ will find itself in- PIGA maps to the X chromosome (4, 5). Its location on the X frame 39 of the translation start of the Piga gene and therefore chromosome explains why a single mutation is sufficient to will be driven by the endogenous Piga promoter. The structure abrogate the expression of GPI-linked proteins. Because of X of wild-type (wt) Piga, lox-Piga-lacZ, and Lox-DPiga-lacZ are chromosome inactivation in female cells, both male and female shown in Fig. 1A. ES cells carrying the lox-Piga-lacZ gene were cells contain one single active Piga gene. In PNH, the mutation injected into C57BL/6 blastocysts to generate chimeric mice. is thought to occur in a hematopoietic stem cell, because Chimeric males were crossbred with C57BL/6 female mice. GPI-deficient cells are found in all blood cell lineages (for Lox-Piga-lacZ showed the expected X linked transmission review, see ref. 6). Although the mutations account for the pattern. Hemizygous lox-Piga-lacZ males were obtained in the deficiency of GPI-linked proteins on the affected blood cells, N2 generation. it is not clear that mutations of the PIGA gene cause the clonal FVB/NJ mice homozygous for the EIIa-cre transgene (EIIa- expansion that enables GPI-anchor-deficient blood cells to cre(1/1) mice) were generously provided by Heiner Westphal become the dominant blood cell population in patients with (National Institutes of Health, Bethesda, MD; ref. 13). Lox- PNH. Piga-lacZ mice were crossbred with EIIa-cre(1/1) mice as GPI-linked proteins are found in almost every tissue and indicated. The lox-Piga-lacZ/EIIa-cre offspring therefore had a serve many different functions (7). An inherited form of mixed genetic background. complete GPI-anchor deficiency has not been reported and is probably not compatible with life. Conforming to the evolu- This paper was submitted directly (Track II) to the Proceedings office. tionary conservation of synteny of the mammalian X chromo- Abbreviations: PIGA, phosphatidylinositol glycan class A; PIGA refers to the human gene, Piga to the murine gene, and PIGA to the gene The publication costs of this article were defrayed in part by page charge product of either species; ES, embryonic stem; PNH, paroxysmal nocturnal hemoglobinuria; GPI, glycosyl phosphatidylinositol; wt, wild payment. This article must therefore be hereby marked ‘‘advertisement’’ in type. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed. e-mail: mbessler@ PNAS is available online at www.pnas.org. im.wustl.edu. 7479 Downloaded by guest on September 25, 2021 7480 Medical Sciences: Keller et al. Proc. Natl. Acad. Sci. USA 96 (1999) 0.7% agarose gels at 38 V for 60–72 hours and transferred to Hybond-N nylon membranes (Amersham Pharmacia) by using Southern blotting. The filters were hybridized to a SacII– BamHI DNA probe including exon 1 (Sac-Bam). The band intensity ratios were defined with a phosphoimager (Molecular Imager System GS-525, Bio-Rad) by using MOLECULAR ANA- LYST version 2.1.2 software (Bio-Rad). Analysis of GPI-Anchored Membrane Proteins. GPI-linked surface proteins on erythrocyte membranes were measured by using flow cytometry (FACscan, Becton Dickinson) with FITC- conjugated mAbs against CD24 (M1/69, PharMingen) (10). RESULTS Production of Mice with High Level of Piga Gene Recom- bination. To obtain high efficiency of Cre-mediated Piga gene recombination, we took advantage of the expression pattern of the EIIa promoter. Expression from the EIIa promoter in the absence of its natural transcriptional activator, the E1A gene product, is restricted to oocytes and preimplantation embryos (15). Therefore, we predicted that Cre-mediated Piga gene recombination in the early embryo will be most efficient if EIIa-cre is maternally derived. Accordingly, we crossbred hemizygous lox-Piga-lacZ(1) males with homozygous EIIa- cre(1/1) females. Thirty-one mice were born. Nineteen were FIG.1.(A) Genomic structure of wt Piga, lox-Piga-lacZ, and males, which, because of the breeding strategy, did not inherit lox-Piga-lacZ after recombination (lox-DPiga-lacZ). Two loxP sites (open arrows) and the coding region for lacZ (lacZ-box) were intro- a lox-Piga-lacZ gene. Twelve offspring were females, and all duced into the Piga locus by homologous recombination in murine ES carried a lox-Piga-lacZ and an EIIa-cre gene. The efficiency of cells (12). In the lox-Piga-lacZ configuration, PIGA function is not Cre-mediated Piga gene recombination in female offspring impaired (filled boxes), and LacZ is not expressed (open lacZ box). varied. Three had, in virtually all cells, a recombined lox-Piga- However, after Cre-mediated excision of the DNA sequences between lacZ gene (highly recombined animals), as verified by the the two lox sites Piga becomes inactivate (open Piga boxes) and LacZ absence of a lox-Piga-lacZ allele determined by PCR of tail falls under the endogenous Piga promoter and is expressed (filled lacZ DNA and Southern blot analysis (see below). In the remaining box). Restriction sites used in the methylation assay are shown. P, PstI; nine females, the extent of Cre-mediated lox-Piga-lacZ recom- S, SacII. Genomic DNA was digested with PstI (P) to obtain restriction bination varied between 0 and 95% (partially recombined fragments suitable for Southern blotting. Each PstI fragment has a different size depending on whether it is derived from the wt (7.0 kb), mice). Thus, the site-specific DNA recombination approach the lox-Piga-lacZ (10.4 kb), or the lox-DPiga-lacZ (8.1 kb) gene. To proved successful in generating female mice that had high- determine the extent of methylation DNA was subsequently digested efficiency Cre-mediated Piga gene recombination.

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