Figure 2

Techniques for manipulating the rat genome. (a) The mutagenicity of N-ethyl-N-nitrosourea (ENU) is the result of the ability to transfer the ethyl group, shown highlighted in orange, to nucleotides in DNA. During replication this can result in the mis-insertion of a nucleotide and after another round of replication in a single base pair substitution. (b) Schematic overview of germline Sleeping Beauty (SB) transposition. A transgenic rat expressing the transposase gene is crossed with a transgenic rat that carries the transposon in its genome. This will produce double transgenic 'seed rats' with transposition events in their germ line, which can be fixed by outcrossing them with wild-type animals. Inverted terminal repeats (ITR) are shown as red triangles. (c) A DSB is introduced at a specific locus by fusing two zinc-finger (ZF) arrays to monomeric FokI domains. When no homologous template is available for repair by homologous recombination, the DSB is repaired by the error-prone mechanism of nonhomologous end joining (NHEJ). This can result in insertions or deletions and consequently out-of-frame mutations. (d) Schematic representation of gene targeting by homologous recombination. A DSB near a gene of interest (G) is repaired using exogenous DNA as template. Black lines indicate DNA sequence homologous to the target; red lines indicate nonhomologous DNA (*).