Sleeping Beauty transposon system-based applications for mouse germline mutagenesis

Investigations into the utility of the Sleeping Beauty (SB) transposon system as an insertional mutagen in the germline of mice have led to several key observations about its activity in mouse chromosomes. The discovery that this fish transposon is highly active in the germline of transgenic mice, the observation that transposition from a chromosomal donor site frequently results in local hopping, the development of methods for rapidly and precisely identifying transposon insertion sites, and the identification of insertional mutations in genes caused by specially-designed mutagenic SB transposon vectors have prepared the SB system to be put to the test as a tool that can contribute to the functional annotation of the mouse genome. This thesis covers three studies that have attempted to advance this technology. The first study describes the systematic development and testing of an advanced SB transposon-based vector system. Significant progress was made developing a "gene-trap tTA" vector system for mutating mouse genes and creating a resource of mouse strains that will allow for conditional expression of genes for functional and genetic studies. The second study used this mutagenic transposon system to explore a new approach for insertional mutagenesis in the mouse. Previous studies had used reverse genetic studies where transposon mobilization, insertion, and resulting mutations were identified using molecular techniques and followed to learn what their consequences were. We anticipated that in order for the SB transposon system to have a major impact on the annotation of the mammalian genome it needs to be tested in the context of a forward-genetic screen. Thus, we performed the first forward-genetic screen for recessive lethal and other visible or behavioral phenotypes using an insertional mutagen. This study was successful in identifying visible, behavioral, and lethal phenotypes and identified other types of transposon-induced mutations in the mouse germline. The final study describes experiments designed to understand the role of cytosine-phosphodiesterguanine (CpG) methylation on the enhancement of SB transposition. In addition, we demonstrate that artificial methylation of transposon substrates can enhance transposition-mediated germline transformation of mice. Taken together, these studies have advanced the field of SB transposon-based applications for mouse functional genomics.