C57BL/6-Klk4^tm1.1Jpsi/J

Stock number: 010684
Research areas: Developmental Biology Research, Internal/Organ Research, Research Tools

Description

These Klk4-null/lacZ knock-in (Klk4^- or Klk4^lacZ) mice harbor a nuclear-localized β-galactosidase "knock-in" mutation that also abolishes endogenous Klk4 (kallikrein related-peptidase 4 [prostase, enamel matrix, prostate]) gene function. These mice may be useful for studying the molecular mechanisms of enamel protein cleavage necessary for proper dental enamel formation/biomineralization/crystallization during tooth development.

Detailed description

Mice homozygous for the Klk4-null/lacZ knock-in allele (Klk4^- or Klk4^lacZ) are viable and fertile, with a nuclear-localized β-galactosidase (NLS lacZ) translation initiation site and coding region replacing the Klk4 translation initiation site and coding region. This abolishes endogenous gene function. Under control of the upstream promoter/enhancer elements, lacZ expression is directed to ameloblasts (the enamel-forming cells in developing teeth) during the transition and maturation stages of dental enamel formation. Klk4-deficiency results in improper dental enamel matrix biomineralization, altered gross tooth morphology, and premature tooth decay. Homozygous mice exhibit malformed enamel that is rapidly abraded after weaning (even when fed moistened chow) and a tendency for all teeth to fracture at points of contact. Heterozygous mice appear indistinguishable from wildtype mice. These Klk4^lacZ mice may be useful for studying the molecular mechanisms of enamel protein cleavage necessary for proper dental enamel formation/biomineralization/crystallization during tooth development.

NLS lacZ activity may be readily distinguished from native mouse lacZ activity by its nuclear localization and high pH optima: mouse β-galactosidase is a lysosomal enzyme that is only marginally active at pH 7.5, whereas bacterial β-galactosidase can be strongly detected above pH 8 (Weiss et al, 1999 Histochem J 31:231).

Development

A targeting vector was designed to insert a nuclear-localized β-galactosidase (NLS lacZ) gene and loxP-flanked PGK-Neo cassette into the first coding exon (exon 2), and also delete the entire coding region (exons 2-6), of the kallikrein 4 gene. This replaces the Klk4 translation initiation site and coding region with the NLS lacZ translation initiation site and coding region. The construct was electroporated into C57BL/6-derived Bruce4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and male chimeras were mated to C57BL/6J females. Next, mutant mice were bred to "B6 Cre deleter" mice (coisogneic on C57BL/6) to remove the floxed neo cassette. The resulting mice harboring the Klk4-null/lacZ knock-in allele (Klk4^- or Klk4^lacZ) were selectively bred to remove the Cre transgene and then backcrossed to C57BL/6J mice for at least six generations prior to arrival at The Jackson Laboratory. Upon arrival, mutant mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

Allele

Symbol: Klk4^tm1.1Jpsi
Name: targeted mutation 1.1, James P Simmer
MGI accession ID: MGI:4355346
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Klk4
Marker name: kallikrein related-peptidase 4 (prostase, enamel matrix, prostate)
Chromosome: 7
Strain of origin: B6.Cg-Thy1^a
Expressed genes: lacZ,beta-galactosidase,E. coli
Site of expression: lacZ expression is directed to ameloblasts (the enamel-forming cells in developing teeth) during the transition and maturation stages of dental enamel formation.
Molecular note: The genomic sequence spanning the initiation codon in exon 2 through exon 6 was replaced with an NLS-lacZ and floxed neo cassette. The neo cassette was subsequently removed by germ line, cre mediated recombination. This allele lacks the entire coding region.