The murine stanniocalcin 1 gene is not essential for growth and development

Abstract

The stanniocalcin 1 (STC1) gene is expressed in a wide variety of tissues, including the kidney, prostate, thyroid, bone, and ovary. STC1 protein is considered to have roles in many physiological processes, including bone development, reproduction, wound healing, angiogenesis, and modulation of inflammatory response. In fish, STC1 is a hormone that is secreted by the corpuscles of Stannius and is involved in calcium and phosphate homeostasis. To determine the role of STC1 in mammals, we generated Stc1-null mice by gene targeting. The number of Stc1-/- mice obtained was in accordance with Mendelian ratios, and both males and females produced offspring normally. No anatomical or histological abnormalities were detected in any tissues. Our results demonstrated that Stc1 function is not essential for growth or reproduction in the mouse.

FIG. 1.

Mouse Stc1 locus, targeting vector, and targeted allele. The upper line shows a map of the Stc1 locus with the four exons defined by the black hatched boxes. The XbaI (X) and NheI (N) restriction sites, protein initiation (atg) and termination (taa) codons are also indicated. The 3′ (P1) and 5′ (P3) external probes used for Southern blot analyses are shown. The diagnostic XbaI restriction fragments (5.1 kb and 3.7 kb) and NheI restriction fragments (7 kb and 4 kb) are shown with broken arrows.

FIG. 2.

Southern blot analysis of wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) mice. (A) Tail DNA was digested with XbaI, transferred onto a BiodyneB membrane, and hybridized with radioactive probe P1. The predicted bands are seen for each genotype. (B) The membrane was stripped and rehybridized with a 0.3-kb cDNA probe specific for Stc1 exons 2 and 3. No band is seen in the −/− lane, indicating targeting of the Stc1 allele. (C) DNA was digested with NheI and hybridized with probe P3. The positions of DNA molecular size markers are indicated at the sides of the blots.

FIG. 3.

Northern blot analysis of wild-type (+/+) and homozygous mutant (−/−) mice. (A) Ten micrograms of total RNA from ovaries was subjected to electrophoresis in a 1% formaldehyde gel, transferred to a Pall BiodyneB membrane, and hybridized with a 0.3-kb cDNA probe that was specific for exon 2 and exon 3. The Stc1 transcript is seen only in the wild-type mice. (B) The blot was hybridized with a 3.2-kb exon 4-specific probe. While the correct transcript is seen in wild-type mice, an extraneous 4-kb band is seen in Stc1^−/− mice. (C) The blot is a duplicate of that in panel B, but it was hybridized instead with a 2-kb Neo-specific probe. This probe hybridized to the novel 4-kb band, suggesting an aberrant splice Neo-Stc1 exon 4 product. The lower 1.4-kb band is the usual Neo transcript. (D) RNA from ovaries isolated from wild-type and Stc1^−/− L278nd mice (that had the Neo cassette deleted) were hybridized with Stc1 cDNA. The novel 4-kb band is no longer present in the L278nd Stc1^−/− mice. The lower panel shows hybridization to GAPDH as a loading control.

FIG. 4.

Sequence analysis of the novel transcript from the targeted allele. (A) The positions of the two RT-PCR primers A1216 and A1252 and the sequencing primer A1218 are indicated. The thicker line below represents the spliced product of the Neo transcript and exon 4, with the intervening intron 3 region deleted. (B) DNA sequence of the Neo-exon 4 spliced product obtained with primer A1218. The italicized letters show sequence derived from the neomycin cassette, and the protein-coding sequence for the neomycin resistance gene is underlined with the termination codon in bold type. The nonitalicized letters are sequence derived from exon 4 of Stc1; the exon 4 sequence is out of frame in the transcript, and there are three additional stop codons (bold type).

FIG. 5.

Western blot analyses of STC1 protein from wild-type and Stc1^−/− mice. (A) Total protein samples (30 μg) from quadriceps muscle of two wild-type (+/+) and two Stc1^−/− (−/−) mice were resolved by SDS-PAGE and immunoblotted with STC1 polyclonal antibody. The leftmost lane contains 10 μg of purified human recombinant STC1 (rSTC1) protein produced by a baculoviral expression system (12). (B) Protein samples (30 μg) from heart (ht) and kidney (kid) were loaded. Cross-reactivities of this antibody are indicated by the presence of various bands that are larger than the STC1 bands in both wild-type and Stc1^−/− tissues. Blots were stained for total proteins with Ponceau S (PonS) to indicate equal loads before immunoblotting was carried out. The positions of molecular mass markers (in kilodaltons) are indicated on the right.

FIG. 6.

Postnatal growth rate. The weights of female and male littermates were measured weekly after weaning (week 1). There were seven females of each genotype, seven Stc1^+/− males, and six Stc1^+/+ and Stc1^−/− males. Error bars indicate standard errors of the means.

FIG. 7.

Measurement of serum calcium and phosphorus concentrations (conc.) in wild-type and homozygous mutant mice. Wild-type and homozygous mutant adult female mice were given intraperitoneal injections of vitamin D3 at a dose rate of 2 μg/kg of body weight daily for 4 days. A small amount of blood was collected from the tail before the start of injections, and terminal blood was taken the morning after the last injections. Bars show mean values, and error bars indicate standard deviations.

FIG. 8.

Analysis of Stc2 RNA expression. RT-PCR amplification of STC2 from various organs isolated from male (A) and female (B) wild-type (+/+) and homozygous mutant (−/−) mice. Amplification of GAPDH was used as the internal control, with a smaller amount of starting cDNA and fewer cycles of amplification. Kid, kidney; Ht, heart; Liv, liver; Lg, lung; Tes, testis; Ov, ovary; Ut, uterus.