To establish a model for oncogenic translocations, we developed an approach in which DSBs are introduced into nonhomologous sequences on murine chromosomes 14 and 17 () (11
), using modifications of our earlier models (12
). DSBs occur in these chromosomes through the expression of the rare-cutting endonuclease I-Sce
I, which efficiently cleaves an 18-bp recognition sequence that has been introduced into mammalian cells (15
). (Mis)repair of the two DSBs by NHEJ involving the two chromosomes leads to a translocation that can reconstruct a functional gene that confers resistance to a neomycin analogue, allowing for selection.
Figure 1 Modeling oncogenic translocations in murine cells. Approach for inducing and recovering translocations resulting from nonhomologous end-joining (NHEJ) between heterologous chromosomes. The reporter contains a neo gene split by an intron into neoSD and (more ...)
More precisely, the translocation selection is based on a neo
gene that has been split by an intron to form neoSD
(containing a splice donor) and SAneo
(containing a splice acceptor) () (12
). The neoSD
gene fragments each contain an I-Sce
I–recognitio1n site that defines the border of their intronic sequences. The gene fragments are targeted to loci on chr.17 and chr.14, respectively, in mouse embryonic stem (ES) cells, given the ease of targeting these cells (17
). Cleavage by I-Sce
I, followed by NHEJ to join neoSD
, results in a translocation that forms a neo+
gene on der17 (). Because the DSBs occur within intronic sequences, a variety of breakpoint junctions are recoverable with neo+
selection, although the maximal deletion that is allowable to recover a neo+
gene is the size of the intron, which here is 2.7 kb. Formation of der(14) is not under constraint.
To induce translocations, I-SceI is transiently expressed in ES cells containing the targeted gene fragments and then the cells are plated. After 48 hours the cells are exposed to the neomycin analogue G418. Within 6 days individual, G418-resistant clones are visible as colonies; these colonies are isolated and expanded. Translocations are evaluated by polymerase chain reaction (PCR), sequencing, Southern blot, and/or fluorescence in situ hybridization (FISH) with chromosome-specific paints. The frequency of translocation formation is calculated as the number of G418-resistant colonies divided by the number of transfected cells.
After expression of I-Sce
clones were recovered at a frequency of ~3 × 10−5
). Recombinants were confirmed to contain the reciprocal t(14;17) using FISH. The high frequency of translocations suggests that endonuclease expression is particularly favorable for generating concurrent DSBs. I-Sce
I cleaves mammalian chromosomes very efficiently (16
). Both sister chromatids may be cleaved by I-Sce
I concurrently, precluding repair by HR between sister chromatids and permitting DNA ends to participate in other repair events. Moreover, if a single DSB is repaired by precise ligation, the I-Sce
I site is restored, offering the potential for multiple cycles of cleavage that could result in translocation formation. The frequency of precise ligation after I-Sce
I expression is not quantifiable in these experiments; only events that disrupt the I-Sce
I site can be scored.