Table 1.

DSB-induced allelic recombination and end-joining in mouse ES cells

Transfected DNA	No. G418R	No. analyzed	No. EJ or STGC	No. LTGC (+NH)	No. duplications
K cell line
pPGK3xnlsI-SceI	13	12	12 (EJ)	0	1
pgklacZ	0
L cell line
pPGK3xnlsI-SceI	0
pgklacZ	0
KL cell line
pPGK3xnlsI-SceI	25	21	16 (EJ/STGC)	5 (3)	1
pgklacZ	0
M cell line
pPGK3xnlsI-SceI	9	8	8 (EJ)	0	0
pgklacZ	0
MN cell line
pPGK3xnlsI-SceI	40	38	26 (EJ/STGC)	12 (1)	13
pgklacZ	0

Approximately 1.6 × 10⁷ cells for each cell line were electroporated with the I-SceI endonuclease expression vector pPGK3xnlsI-SceI or, as a mock DNA control, pgklacZ and selected in G418. Correcting for 50% cell killing, the frequency of neo⁺ colonies was 1.1–1.6 × 10⁻⁶ (K and M cell lines) or 3.0–4.8 × 10⁻⁶ (KL and MN cell lines). Allelic recombination with LTGCs in the KL and MN cell lines was identified by Southern blot analysis (Fig. 2). Some LTGC clones displayed altered bands consistent with coincident nonhomologous (+NH) events, the number of such clones being indicated in parentheses. K and M alleles undergo nonhomologous DNA end-joining (EJ) at the Sneo gene to give rise to G418^R clones. In addition, in the KL and MN cell lines, the K and M alleles can also undergo allelic recombination with STGCs, due to the presence of the L and N alleles, respectively. EJ and STGC cannot be distinguished. Duplications of the Rb locus were also detected in some of the G418^R clones, as identified by additional bands and/or increased hybridization intensity (Figs. 2 and 3).