Since the discovery of calbindin D9k, its role in intestinal calcium absorption has remained unsettled. Further, a wide distribution of calbindin D 9k among tissues has argued for its biological importance. We discovered a frameshift deletion in the calbindin D 9k gene in an ES cell line, E14.1, that originated from 129͞OlaHsd mice. We produced mice with the mutant calbindin D9k gene by injecting the E14.1 ES cell subline into the C57BL͞6 host blastocysts and proved that these mice lack calbindin D 9k protein. Calbindin D9k knockout mice were indistinguishable from wild-type mice in phenotype, were able to reproduce, and had normal serum calcium levels. Thus, calbindin D 9k is not required for viability, reproduction, or calcium homeostasis.vitamin D 3 ͉ E14ES cells ͉ calcium homeostasis F or decades, a major physiologic function of calbindin D 9k was believed to be a carrier of calcium during intestinal calcium absorption (1). However, the detailed mechanism of vitamin D-induced intestinal calcium absorption still is not fully understood. According to the currently accepted model, vitamin D-mediated transcellular calcium absorption in the intestine proceeds through the calcium channel proteins TRPV5 and TRPV6 (2) with the involvement of cellular calcium transfer protein calbindin D 9k (1, 3) and a calcium extrusion protein, calcium ATPase (PMCA 1b ) (4). The data on regulation of epithelial calcium channels (TRPV5 and TRPV6), calbindin D 9k , and PMCA 1b expression by 1,25-dihydroxyvitamin D 3 seem clear, and these genes have vitamin D responsive elements in their promoter regions (5-7).Calbindin D 9k was first discovered as the mammalian counterpart to the calbindin D 28k that was found in chick duodenal mucosa in response to vitamin D 3 (8, 9). Later, calbindin D 9k from vitamin D 3 -responsive rat intestinal mucosa was identified, purified, and characterized (9, 10). Since its discovery in 1967, the role of calbindin D 9k protein in vitamin D 3 -mediated intestinal calcium absorption has been intensively studied but still remains unsettled. In the meantime, a wide distribution of this protein in tissues not involved in calcium absorption has been noted, arguing for its importance in biology (11-13).In 1969, Harmeyer and DeLuca (14)
Results and DiscussionDiscovery of the Mutant Mouse Calbindin D9k Gene. The P1 clone 7681 with an 85-kb insert of genomic DNA͞SauIIIA containing the mouse calbindin D 9k gene was purchased from Incyte Genomics (Palo Alto, CA). The P1 mouse library was generated by Sternberg et al. (20) by using genomic DNA from the ES cell line subclone E14.1, which is derived from E14 ES cells (21). The ES cell line E14 was derived from the inbred mouse strain 129͞OlaHsd in 1985 by Hooper et al. (22). The insert was cut with BamHI and subcloned into pBluescript (Stratagene, La Jolla, CA). The clone that was positive for the calbindin D 9k gene was selected by Northern blot analysis, and a plasmid with the mutant calbindin D 9k insert was isolated. We have sequenced the 10.409-kb insert containi...