X inactivation and somatic cell selection rescue female mice carrying a Piga-null mutation

Abstract

A somatic mutation in the X linked PIGA gene is responsible for the deficiency of glycosyl phosphatidylinositol (GPI)-anchored proteins on blood cells from patients with paroxysmal nocturnal hemoglobinuria. No inherited form of GPI-anchor deficiency has been described. Because conventional Piga gene knockout is associated with high embryonic lethality in chimeric mice, we used the Cre/loxP system. We generated mice in which two loxP sites flank part of Piga exon 2. After crossbreeding with female mice of the EIIa-cre strain, the floxed allele undergoes Cre-mediated recombination with high efficiency during early embryonic development. Because of X chromosome inactivation, female offspring are mosaic for cells that express or lack GPI-linked proteins. Analysis of mosaic mice showed that in heart, lung, kidney, brain, and liver, mainly wild-type Piga is active, suggesting that these tissues require GPI-linked proteins. The salient exceptions were spleen, thymus, and red blood cells, which had almost equal numbers of cells expressing the wild-type or the recombined allele, implying that GPI-linked proteins are not essential for the derivation of these tissues. PIGA(-) cells had no growth advantage, suggesting that other factors are needed for their clonal dominance in patients with paroxysmal nocturnal hemoglobinuria.

Figure 1

(A) Genomic structure of wt Piga, lox-Piga-lacZ, and lox-Piga-lacZ after recombination (lox-ΔPiga-lacZ). Two loxP sites (open arrows) and the coding region for lacZ (lacZ-box) were introduced into the Piga locus by homologous recombination in murine ES cells (12). In the lox-Piga-lacZ configuration, PIGA function is not impaired (filled boxes), and LacZ is not expressed (open lacZ box). However, after Cre-mediated excision of the DNA sequences between the two lox sites Piga becomes inactivate (open Piga boxes) and LacZ falls under the endogenous Piga promoter and is expressed (filled lacZ box). Restriction sites used in the methylation assay are shown. P, PstI; S, SacII. Genomic DNA was digested with PstI (P) to obtain restriction fragments suitable for Southern blotting. Each PstI fragment has a different size depending on whether it is derived from the wt (7.0 kb), the lox-Piga-lacZ (10.4 kb), or the lox-ΔPiga-lacZ (8.1 kb) gene. To determine the extent of methylation DNA was subsequently digested with the methylation-sensitive restriction endonuclease SacII (S). If the 5′ SacII site is unmethylated, each fragment is shortened by 0.5 kb. The Sac-Bam DNA probe used for hybridization is shown. (B) The diagram shows the promoter region and intron 1 of the Piga gene with the endonuclease cleavage sites that are subject to methylation depending on the inactivation status of the gene. Lower shows the proportions of DNA that is methylated in wt mice. Values were defined by the use of a phosphoimager (see Materials and Methods) and are shown as the mean and SD of all measured samples. n.a., not analyzed.

Figure 2

Southern blot analysis to determine the transcriptional activity of the Piga alleles. (A) Southern blot analysis of DNA obtained from wt animals and mice hemi-, hetero-, or homozygous for the lox-Piga-lacZ gene. (B) Southern blot analysis of DNA isolated from different organs of a female mouse with a lox-ΔPiga-lacZ gene in almost all cells (mouse E, Fig. 3B). DNA was digested with PstI alone (P) or with PstI and SacII (PS). Southern blots were hybridized with the Sac-Bam DNA probe. The 10.4-kb fragment corresponds to lox-Piga-lacZ, the 8.1-kb fragment to lox-ΔPiga-lacZ, and the 7.0-kb fragment to the wt Piga gene. Digestion of the nonmethylated portion with SacII shortens each restriction fragment by 0.5 kb. Sp, spleen; Th, thymus; Li, liver; K, kidney; L, lung; B, brain. F (father) displays DNA of a male lox-Piga-lacZ(+) mouse, M (mother) shows DNA of a female EIIa-cre(+/+) mouse.

Figure 3

Diagram summarizing methylation at the 5′ SacII restriction site in different tissues. (A) Methylation of wt and lox-Piga-lacZ in 3 mice heterozygous for lox-Piga-lacZ. (B) Methylation of wt and lox-ΔPiga-lacZ in 3 mice that had high levels of Piga gene recombination. The Southern blot analysis of mouse E is shown in Fig. 2B. DNA samples from the heart of mouse A and E and from the liver of mouse A and C were degraded. DNA from heart, lung, kidney, and spleen of mouse D were lost accidentally.

Figure 4

Proportion of PIGA-deficient red blood cells in mice with a lox-ΔPiga-lacZ gene in almost every cell. Flow cytometric analysis of peripheral red blood cells obtained from mice D–F and from an age-matched lox-Piga-lacZ control mouse were stained with FITC-conjugated mAb against the GPI-linked antigen CD24 and analyzed by flow cytometry.

Figure 5

Negative correlation of the level of DNA methylation and the extent of lox-Piga-lacZ recombination. The plot displays the percentage of methylation at the 5′ SacII site of the wt gene (y axis) as a function of the amount of recombination of the lox-Piga-lacZ gene (x axis). Shown are the regression line and the 95% confidence interval. r, correlation coefficient.