Generation of knockout rats by insertional mutagenesis with gene trap transposons in spermatogonial stem cells. Cultured stem cells are transfected with gene trap transposon and transposase constructs that will lead to thousands of transposon insertions covering all chromosomes. Those cells in which insertions have occurred in expressed genes can be selected based on activation of the gene trap marker, and the insertion sites can be mapped. Cell clones or polyclonal insertion libraries can be transplanted into the testes of sterile males, in which the spermatogonial step cells will undergo spermatogenesis. These transplanted males are crossed with wild-type females to pass the insertions through the germline and generate transgenic/knockout animals.