Generation of a Kallikrein 4 Knockout/LacZ Knockin Mouse

KLK4 is a serine protease with broad target sequence specificity that is thought to degrade accumulated enamel matrix proteins, facilitating their removal and replacement by mineral, which is essential for enamel hardening. In humans, KLK4 mutations cause autosomal recessive hypomaturation amelogenesis imperfecta. The enamel is less mineralized and is pigmented owing to failure of mineral to replace organic matrix. Objectives: To better understand Klk4 function and to better characterize its normal temporal and spatial patterns of expression, we have used gene targeting to knock out normal Klk4 expression, while replacing the 5' Klk4 coding sequence with a ß-galactosidase reporter in mice. Methods: The Klk4 5' arm in the targeting vector ended just upstream of the native Klk4 translation initiation codon and was joined to a lacZ (ß-galacatosidase) reporter with a mouse nuclear localization signal (NLS). Successful homologous recombination of the targeting construct in ES cells that survived G418/gancyclovier positive-negative selection was determined by Southern blot hybridization. A positive clone was expanded and the resultant chimera was mated to C57BL/6 mice and the offspring were analyzed for germ line transmission by Southern blot hybridization. Heads from heterozygous (Klk4^+/lacZ) mice at ages 4, 8, and 12 days were fixed in paraformaldehyde, processed for ß-galactosidase staining, counterstained with hematoxylin, and observed by light microscopy. Results: ß-galactosidase staining of mouse sections shows intensive signal over maturation stage ameloblasts. No signal is observed in secretory stage ameloblasts, odontoblasts, or any other tissue in the head. These results correlate well with previous studies of Klk4 expression by in situ hybridization, excepting the lack of expression by odontoblasts. Conclusions: We have generated a Klk4 knockout/lacZ knockin mouse that will permit better characterization of Klk4 expression and function. This study is supported by NIDCR grants DE011301 and DE015846.