Beta Defensins and Immunological Homeostasis in the Mouse

Defensins are a group of proteins produced by some white blood cells, skin cells and the cells lining mucosal surfaces of the body. They have the unique ability to directly kill harmful bacteria and thus are a first line of defence of the organism. Based on their structure defensins are grouped in three families: alpha, beta, and theta defensins. Several defensin molecules have been well characterised, but there are many others whose existence was recently discovered, and whose role for the preservation of organism's defensive equilibrium (immunological homeostasis) remains unclear. Moreover, recent evidence suggests that defensins may have additional, diverse functions in addition to the ability to kill bacteria. Using an advanced technology for gene manipulation called oligo targeting, we were recently able to 'switch off' the MBD14 gene and created a new mouse model for beta-defensin deficiency. In this proposal we would like to refine this technology further and apply it for the inactivation of two other beta defensin genes with unknown functions. In addition, we would like to investigate the generated MBD14 mutant and the newly generated mice and describe the effects of these manipulations on organism's defence systems. The outcome of this project will increase our understanding about the role of beta defensins in immunological homeostasis.

Defensins are a group of anti-bacterial peptides produced by some white blood cells, skin cells and the cells lining mucosal surfaces. They play a major role in innate immunity by directly killing bacteria. Based on their structure defensins are grouped in three families: alpha, beta, and theta defensins. Several defensin molecules have been well characterised, but there are many others whose existence was recently discovered, and whose role for the preservation of immunological homeostasis remains unclear. Moreover, recent evidence suggests that defensins may have additional, diverse functions besides the ability to kill bacteria and are promising candidates for anti-bacterial therapy. Beta defensin genes in the mouse are spread in several clusters on chromosomes 1, 2, 8 and 14. Using an advanced technology for gene manipulation called oligo targeting, we were recently able to inactivate the mouse beta defensin 14 gene (MBD14, Defb14) and created a new model for beta-defensin deficiency. Oligo targeting is a fast and relatively simple approach for gene inactivation based on ES cells of the 129 strain where the mismatch repair protein MSH3 was inactivated. It allows targeting of neighbouring genes situated in clusters, which makes it particularly suitable for the manipulation of defensin genes. Here we would like refine the technique further by developing an RNAi based MSH3 knockdown in ES cells so it can be used with ES cells of any mouse strain. We will apply this approach to target the entire defensin cluster in chromosome 1 and generate mutants where Defb18 and Defb41 have been inactivated . In addition, we will investigate the in vivo effects of beta defensin inactivation in the MBD14 mutant and the newly generated mutant mice using a comprehensive phenotyping protocol for assessment of mouse immunity. The outcome of this project will increase our understanding about the role of beta defensins in immunological homeostasis.