EXCEED THE SPACE PROVIDED. Insertional mutagenshave been particularlyvaluablefor geneticists. Inthe mouse,the useof insertionalmutagenesis for germlinegeneticscreens hasbeen limitedto transgeneinsertionand gene trapping inembryonicstem cell lines. In this proposal,we further characterizeand developa methodfor invivo gene trappingin the mouseusinga transposableelement. The methodrelieson the useof a synthetictransposableelementcalled Sleeping Beauty(SB), which is a reconstructedmemberof the Tcl/mariner family of DNA transposableelements. We havedemonstratedthat efficient mobilizationof SB transposonscan be made to occurin the germlineof transgenic miceand that noveltransposon insertionscan occurin or neargenes. Wepropose optimizationof the SB systemfor germlineand somatic cellinsertionalmutagenesisinthe mouse. Our goals includeadditional improvementsinthe transposonvectorand the transposaseenzyme, someof which havealreadybeen madeand are nowbeingtested mice, andsomeof which willbe achieved usinga novelselection methodthat utilizesthe retrovirallife cycle. Wewill determinehowthe siteof transposon excisionis repaired inthe presenceof a perfect copy of the transposonon a homologous chromosomeand on a geneticbackgroundwhich is defectivefor non-homologousend joining. Wewill also createa system in which ;potentiallyoncogeneictransposonsaremobilizedin somaticcellswith an aimto create modelsof cancerin the mousecaused by transposoninsertional mutagenesis. Finally,we haveshownthat thetransposableelement canbe used for stablelong-term gene transferinto somaticcells of mice andwe proposeto usethis techniqueto create modelsof cancerinthe mouseinwhich oncogenesand putativeoncogenesare directlytransferredinto somatictarget cells. Insummary,this proposalaimsto utilize, further improve,and better understanda mammalianactive transposableelementfor usein mousegermlinegeneticscreens,somatic cell mutagenesisfor cancergenes,and as a gene transfervehiclefor cancerstudies. PERFORMANCE SITE ========================================Section End===========================================

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA014764-03
Application #
6836058
Study Section
Genome Study Section (GNM)
Program Officer
Satterlee, John S
Project Start
2003-02-15
Project End
2006-11-30
Budget Start
2004-12-01
Budget End
2006-11-30
Support Year
3
Fiscal Year
2005
Total Cost
$314,796
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Genetics
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Maroteaux, G; Loos, M; van der Sluis, S et al. (2012) High-throughput phenotyping of avoidance learning inĀ mice discriminates different genotypes andĀ identifies a novel gene. Genes Brain Behav 11:772-84
Carlson, Daniel F; Geurts, Aron M; Garbe, John R et al. (2011) Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition. Transgenic Res 20:29-45
Mashhoon, Yasmin; Janes, Amy C; Jensen, J Eric et al. (2011) Anterior cingulate proton spectroscopy glutamate levels differ as a function of smoking cessation outcome. Prog Neuropsychopharmacol Biol Psychiatry 35:1709-13
Largaespada, David A (2009) Transposon mutagenesis in mice. Methods Mol Biol 530:379-90
Keng, Vincent W; Ryan, Barbara J; Wangensteen, Kirk J et al. (2009) Efficient transposition of Tol2 in the mouse germline. Genetics 183:1565-73
Collier, Lara S; Largaespada, David A (2007) Transposable elements and the dynamic somatic genome. Genome Biol 8 Suppl 1:S5
Geurts, Aron M; Wilber, Andrew; Carlson, Corey M et al. (2006) Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon. BMC Biotechnol 6:30
Collier, Lara S; Largaespada, David A (2006) Transforming science: cancer gene identification. Curr Opin Genet Dev 16:23-9
Geurts, Aron M; Hackett, Christopher S; Bell, Jason B et al. (2006) Structure-based prediction of insertion-site preferences of transposons into chromosomes. Nucleic Acids Res 34:2803-11
Hackett, Perry B; Ekker, Stephen C; Largaespada, David A et al. (2005) Sleeping beauty transposon-mediated gene therapy for prolonged expression. Adv Genet 54:189-232

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