Searching for Genes of Host Defense

Through random mutation of the mouse genome and phenotypic screening of the mutated mice, genes can be identified that are associated with dysfunction in the innate immune system. The strategy proposed works under the knowledge that many genes are involved in the immune system and that random mutation could lead to a change in their genetic code. This mutation can present as a phenotypically abnormal immune system. Once a phenotype is identified, the genome can be analyzed in an attempt to trace the mutated gene responsible for the weakened immune system. One of the elegant aspects of this genetic method is that it does not rely on a hypothesis about how the immune response works. This leads to an unbiased approach where interpretation errors are rarely made. A forward genetic approach is used to create abnormal phenotypes of the innate immune system and then determine the genetic cause. The normal mutation rate is accelerated by the widely used germline mutagen N-ethyl-N-nitrosourea (ENU) to produce an average of 3,000 single nucleotide changes per host leading to an average of 60 coding changes. To produce homozygotes, males of the G1 generation are bred with normal mice of the same strain to yield the G2 generation. Recessive mutations can be found in the G3 generation by a backcross of G2 females with the G1 father. Screening 6 G3 progeny should capture 50% of the mutations in the homozygous form. Phenotypic screening was performed on peritoneal macrophages ex vivo by stimulation with the following toll-like receptor agonists: lipopolysaccharide (TLR4), double stranded RNA (TLR3), triacylated lipoprotein (TLR 1/2), diacylated lipoprotein (TLR 2/6), resiquimod (TLR 7), and unmethylated DNA (TLR 9). The inflammasome pathway was probed by lipopolysaccharide priming followed by stimulation with either nigericin (K+ efflux) or ATP. The secreted TNF-alpha (TLR screen) and IL-1beta (inflammasome screen) were measured by ELISA to determine phenovariance. This research can lead to a deeper understanding of how we combat infection. The study can lead to the development of mutations involved in both the innate and adaptive immune system so autoimmune diseases can also be studied. A long term goal is to identify genes that would render an individual resistant to infection and to study the interaction of these genes.