RESUMO
Kallikrein-related proteases (KLKs) play a critical role in epidermis physiology and have been implicated in skin pathologies such as Netherton syndrome. The contribution of individual KLKs to skin proteolysis is poorly understood. Monitoring of their activities in skin proteome is hampered by overlapping substrate specificities, and there is a need for novel assays. Here, we present a platform of selective and sensitive fluorogenic substrates and inhibitors for profiling KLK5, KLK7 and KLK14. These chemical tools were evaluated using recombinant KLKs and tissue from a unique set of mice deficient in eight combinations of KLKs and their natural regulator LEKTI.
Assuntos
Modelos Animais de Doenças , Calicreínas/deficiência , Calicreínas/metabolismo , Proteólise , Animais , Perfilação da Expressão Gênica , Humanos , Calicreínas/genética , Camundongos , Camundongos Knockout , Pele/metabolismoRESUMO
Netherton syndrome (NS) is caused by mutations in the SPINK5 gene. Several Spink5-deficient mouse models were generated to understand the mechanisms of NS in vivo. However, Spink5-deficiency in mice is associated with postnatal lethality that hampers further analysis. Here we present a viable mouse model for NS generated by mosaic inactivation of the Spink5 gene. We propose that these mice are a valuable experimental tool to study NS, especially for long-term studies evaluating potential therapeutic compounds. Furthermore, we show that mosaic inactivation of a gene using TALENs or CRISPR/Cas9 systems can be used to study lethal phenotypes in adult mice.
Assuntos
Modelos Animais de Doenças , Inativação Gênica , Mosaicismo , Síndrome de Netherton/genética , Serpinas/deficiência , Serpinas/genética , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Inibidor de Serinopeptidase do Tipo Kazal 5RESUMO
Gene targeting in mice mainly employs homologous recombination (HR) in embryonic stem (ES) cells. Although it is a standard way for production of genetically modified mice, the procedure is laborious and time-consuming. This study describes targeting of the mouse Rosa26 locus by transcription activator-like effector nucleases (TALENs). We employed TALEN-assisted HR in zygotes to introduce constructs encoding TurboRFP and TagBFP fluorescent proteins into the first intron of the Rosa26 gene, and in this way generated two transgenic mice. We also demonstrated that these Rosa26-specific TALENs exhibit high targeting efficiency superior to that of zinc-finger nucleases (ZFNs) specific for the same targeting sequence. Moreover, we devised a reporter assay to assess TALENs activity and specificity to improve the quality of TALEN-assisted targeting.