Generation of mouse conditional and null alleles of the type III sodium-dependent phosphate cotransporter PiT-1

Inorganic phosphate (Pi) is required for cells, and as a negatively-charged anion, must be actively transported into the cell. The highly-homologous and ubiquitously-expressed transmembrane type III sodium-dependent inorganic phosphate transporters (PiT) PiT-1 (also called Slc20a1 or Glvr-1) and PiT-2 (also called Slc20a2 or Ram-1) are proposed to have a general “housekeeping” function of transporting Pi into cells (reviewed in Collins, et al., 2004). Inorganic phosphate additionally plays a role in diseases where ectopic calcification occurs, as fluctuations in mineral metabolisms are risk factors. Hyperphosphatemia is correlated with severe coronary calcification in patients with end-stage renal disease (Shigematsu, et al., 2003; Tomiyama, et al., 2006). Studies into the function of PiT-1 uncovered an essential role in human vascular smooth muscle cell calcification, as cells with reduced PiT-1 levels had reduced susceptibility to phosphate-induced calcification (Li, et al., 2006). Supporting a role for PiT-1 specifically in ectopic calcification, a recent study of Werner Syndrome patients with subcutaneous calcification had overexpression of PiT-1 in their skin (Honjo, et al., 2008). Thus, animal models to examine the physiological and potential pathological role of PiT- 1 are of great interest. Since there were no available knockout mice of PiT-1, this is the first report of the creation of conditional (PiT-1flox) and null (PiT-1Δe3,4) alleles in the mouse model. Here we describe the generation of a murine PiT-1 conditional allele with loxP sites flanking exons 3 and 4, allowing for the conditional deletion of these exons by Cre recombinase. The mouse PiT-1 gene consists of eleven exons and spans over 12.8 kb. Ten of these eleven exons code for a protein of 681 amino acid residues and has at least ten transmembrane domains (Boyer, et al., 1998). Amino acid residues critical for Pi transport have been determined for PiT-2 and include D28, E68, S113, D506, E575, and S593 (Bottger and Pedersen, 2002; Bottger and Pedersen, 2005; Salaun, et al., 2004); these sites are conserved in PiT-1 and thus, would also be predicted to be critical for Pi transport. Removal of exons 3 and 4 in the PiT-1 gene results in a shift in the protein reading frame after position 116 and the creation of an early stop codon after position 120; this truncation removes four of the six residues involved in Pi transport and 83% of the full-length protein. Thus, this PiT-1 recombined allele is predicted to be a null allele. To generate the conditional allele of PiT-1, a targeting vector was constructed where a loxP site was placed upstream of exon 3 and a pGK-gb2-neomycin (Neo) resistance cassette flanked by loxP and FRT sites was placed downstream of exon 4, with Neo being in opposite orientation to the PiT-1 reading frame: this is the PiT-1flox-neo allele (Fig. 1a). The targeting vector was linearized and electroporated into iTLBA1 mouse embryonic stem (ES) cells and neomycin-resistant colonies were screened via PCR for confirmation of correct targeting of the short and long homology arms (Fig. 1b and c, respectively). Though a number of ES cell colonies had recombination of the short homology arm (Fig. 1b), not all had recombination of the long arm (clones 112 and 311) (Fig. 1c). Two clones that had recombination of both arms (clones 111 and 221) were selected for microinjection and produced chimera offspring. Germline transmission of the conditional allele PiT-1flox-neo from clone 111 was confirmed by PCR screening (data not shown). Fig. 1 Generation of the PiT-1flox-neo allele. (a) A genomic sequence (shown to scale) containing exons 2-4 was subcloned for targeting vector construction and a loxP site was placed between exons 2 and 3, while a Neo cassette (in reverse orientation) flanked ... The PiT-1+/flox-neo mouse was bred to a FLP1 mouse line to remove the Neo cassette via the FRT sites and generated the PiT-1flox allele in somatic and germ tissue. PiT-1flox heterozygous animals were bred and wildtype (WT), heterozygous, and homozygous progeny were produced at Mendelian ratio (Table 1). PCR analysis using primers that flank the loxP sites were able to detect the 3′ insertion and removal of the Neo cassette leaving the 5′ loxP site and a FRT site (Fig. 2a and b). Mice heterozygous and homozygous for the conditional allele were viable, healthy, fertile, and grossly indistinguishable from wildtype littermates. Both of the loxP sites are functional, as exposure to Cre recombines the allele into the null allele (data not shown). Fig. 2 Alleles from Cre and FLP-mediated recombination and the PCR strategy used to identify them. (a) WT (top), PiT-1flox (middle), and PiT-1Δe3,4 (bottom) alleles (to scale) and relative placement of the primers (arrows) used to genotype them. (b) ... Table 1 Ratio of wildtype (WT), heterozygous, and homozygous offspring at postnatal day (P) 21 from heterozygous matings. The PiT-1+/flox-neo mouse was additionally bred to a Cre mouse line to produce the PiT-1Δe3,4 allele in somatic and germ tissues. PiT-1Δe3,4 heterozygous mice were bred, but only WT and heterozygous progeny were born; the absence of PiT-1 null offspring was statistically significant (p<0.0001) (Table 1). PCR analysis confirmed removal of exons 3 and 4 (Fig. 2a and c) in the heterozygous mice, and they were viable, healthy, fertile, and grossly indistinguishable from wildtype littermates. Because homozygous null mice were not observed at weaning or as neonates, it was necessary to analyze the embryos to confirm that the null progeny were being created, and to determine the cause of lethality. PCR analysis of early embryonic stages confirmed that PiT-1 homozygous null progeny were being produced (Fig. 3a). Semi-quantitative RT-PCR using primers that amplify only exons 3 and 4 did not detect transcript in the null embryos (Fig. 3b, top panel). Primers in exons 2 and 5 amplified a truncated PiT-1 transcript product lacking exons 3 and 4 (exon 2/5) only in tissues that had the null allele, and the larger wildtype product (exon 2-5) in tissues that had a wildtype allele (Fig. 3b, middle panel). Thus, the null embryos do not express wildtype mRNA, only the truncated transcript from the null allele. Fig. 3 Analysis of PiT-1Δe3,4/Δe3,4 during embryonic development. (a) Genotyping for the PiT-1Δe3,4 allele. Primers Ex2F/int4R3/Ex4F amplify the WT allele (268 bp) and the PiT-1Δe3,4 allele (504 bp). Breeding of PiT-1+/Δe3,4 ... Wildtype, PiT-1+/Δe3,4, and PiT-1Δe3,4/Δe3,4 embryos were observed at Mendelian ratios during early embryonic stages (Table 2); after embryonic day (E) 14.5, the absence of PiT-1Δe3,4/Δe3,4 embryos became statistically significant (p=0.02) and thus, the null genotype was lethal past mid-gestation. Analysis of PiT-1Δe3,4 homozygous embryos reveled an abnormal phenotype beginning at E10.5. The embryos all appeared phenotypically normal (Fig. 3c); however, in a portion of the homozygous nulls, a defect in the vasculature system of the yolk sac could be observed (Fig. 3d). These abnormal yolk sacs had a decrease in the superficial vasculature and appeared severely anemic. By E12.5, the PiT-1Δe3,4 homozygous embryos displayed significant growth retardation and anemia as compared to wildtype littermates (Fig. 3e). In addition, an abnormal yolk sac vasculature was observed in all homozygous null yolk sacs at this time point (Fig. 3f). Lack of erythroid cells and a mature vasculature system to bring nutrients and oxygen to the embryo is incompatible with life since the embryos have developed pass the stage where these requirements could have been obtained by diffusion. Thus, we speculate that the anemia and vascular defects in the PiT-1Δe3,4/Δe3,4 embryos leads to hypoxia and nutrient-deprivation, growth arrest, and lethality. Consistent with this, null embryos were observed resorbing at E12.5 (Fig. 3e, far left) and were completely resorbed after E14.5 (Table 2). The observation that these embryos were not resorbing at the same developmental stage could be due, in part, to the mixed strain background these mice were on. All wildtype and heterozygous embryos analyzed were developmentally normal, consistent with observing the expected number of these progeny at weaning (Table 1). Table 2 Ratio of embryos from PiT-1+/Δe3,4 matings at various embryonic (E) stages and the number of abnormal embryos observed. Changes in mRNA expression level of the other type III sodium-dependent phosphate transporter PiT-2 was assayed to determine if PiT-2 expression increased to compensate for the loss of wildtype PiT-1 mRNA in the null progeny. There was no observed increase in PiT-2 transcript when comparing null and wildtype yolk sacs (Fig. 3g, left) or embryos (Fig. 3g, right) at E10.5 (when phenotypes were observed only in the yolk sac) and E11.5 (when phenotypes were observed in both the yolk sac and embryo). These results indicate that loss of exons 3 and 4 in the PiT-1 gene results in a null allele with an embryonic lethal phenotype. PiT-1 is thus necessary for embryonic development, and is non-redundant, as PiT-2 could not compensate for the loss of PiT-1 and allow development into adulthood. The PiT-1 conditional allele provides a useful tool to study the tissue-specific effects of removing a ubiquitous sodium-dependent phosphate cotransporter. Considering the role PiT-1 plays in matrix calcification in vitro, effects on the in vivo calcification of normal bone development can be studied, along with the effects of PiT-1 loss in mouse vascular calcification models. Use of temporal-specific control of Cre expression will allow for the ability to study the function of PiT-1 later in development and in adulthood in the vasculature system.