Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention

Abstract

The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.

Fig. 1.

Simultaneous gene mutation and identification by gene-trapping in mouse ES cells. A retroviral vector contains a splice acceptor sequence (SA) followed by a promoterless selectable marker such as β-Geo, a functional fusion between the β-galactosidase and neomycin resistance genes, with a polyadenylation signal (pA). Insertion of the retroviral vector into an expressed gene leads to the splicing of the endogenous upstream exons (hatched boxes) into this cassette to generate a fusion transcript. The vector also contains a promoter that is active in ES cells [such as that of the mouse phosphoglycerate kinase (Pgk) gene] followed by a first exon (such as that of the Btk gene) upstream of a splice donor (SD) signal. Splicing from this signal to the exons downstream of the insertion gives rise to a fusion transcript that can be used to generate a sequence tag (OST) of the trapped gene by RACE (15). The Btk exon contains termination codons in all reading frames to prevent translation of downstream fusion transcripts.

Fig. 2.

(A) Distribution of OSTs per OmniBank cluster is shown. (B) Historic progression of the estimated genome coverage of OmniBank is shown. (C) Distribution of OSTs relative to the cDNA sequence within the 2,170 genes analyzed is shown. (D) Estimated OmniBank gene coverage by gene family is shown. SEC, secreted; GPR, G protein-coupled receptor; NHR, nuclear hormone receptor; CHA, channel; TRA, transporter; INH, inhibitor; PRT, protease; KNS, kinase; PHD, phosphodiesterase; PHO, phosphatase; ENZ, enzyme; MAS, membrane and secreted; MEM, membrane; SGT, signal transduction; MSC, miscellaneous; RAP, receptor-associated protein; SKL, cytoskeletal; TRF, transcription factor. (E) Distribution of OmniBank gene-trap events throughout the mouse genome. Units on the x axis represent 20-Mbp genomic intervals. The y axis shows the percentage of genes trapped within each 20-Mbp interval. Additional detail on chromosomewide coverage is available in Fig. 6.

Fig. 3.

Generation of Wnk1-deficient mice. Primers A and B flank the genomic insertion site of the gene-trap vector in intron 1 of the Wnk1 gene and amplify a product for the WT allele. The LTR reverse primer, complementary to OmniBank vectors, was used in conjunction with primer B to specifically amplify the mutated allele. Rev, reverse; Mut, mutated.

Fig. 4.

Assessing the mutagenicity of OmniBank gene-trap insertions by RT-PCR. Primers A and B are complementary to exons flanking the insertion site in the PolH gene in mouse chromosome 17 (Chr17) (GenBank accession no. NM_030715). RT-PCR using primers A and B shows the absence of endogenous message in the spleen and thymus of homozygous animals. Control primers to the murine β actin gene were used (GenBank accession no. M12481).

Fig. 5.

Lower blood pressure in heterozygous Wnk1 mice. Blood pressure was measured in untreated WT (n = 20), Wnk1 heterozygotes (n = 11), and WT animals treated with 1.25 mg of the antihypertensive drug enaprilat per kg (n = 3). [Enaprilat is an inhibitor or angiotensin-converting enzyme (ACE), a key regulator of blood pressure in mouse and man.] Blood pressures were measured 10 times per day for 4 consecutive days, and a mean value was generated for each individual mouse. n, Number of mice per cohort.