probe 1

1. 1.5 kb a b 1.04 kb H M H M H M H M D D D D pA MSCV SA SD En2-SA 5985 5993 6044 6057 6065 6070 6072 6113 6132 6136 M H M H M H M H M H M H M H M H M H M H 2.0 kb En2 T2/Onc2 1.04 kb Supplementary Figure 1. Generation and characterization of T2/Onc2 transgenic founders. a, Tail biopsy DNA was digested with DraI, blotted and probed with a fragment of the En2 splice acceptor (blue line). The signal from the 1.5kb transposon fragment was quantified by comparison to the 2.1kb fragment from the En2 locus. b, Tail biopsy DNA from transgenic animals was first digested with DraI then purified and cut with either MspI or HpaII. Genomic CpG methylation of the CCGG recognition sequence will inhibit HpaII but not MspI. The percentage of methylated sites can be determined by comparing the intensities of the 1.04 kb band detected by the probe (blue line) in the MspI and HpaII lanes for each DNA. 7.5 kb 7.4 kb a b probe 1 S ex 1 B S B S probe 2 RosaSB ex 2 neo SB11 probe 1 probe 2 6.6 kb S S B B wt 8.9 kb Supplementary Figure 2. Generation and characterization of RosaSB knock-in allele. a,Structure of the wildtype and RosaSB knockin alleles [FRT sites,(blue triangles), SpeI sites (S), BglI sites (B). b, Southern blotting on tail biopsy DNA shows the predicted fragments and germline transmission of the RosaSB allele. Probe 1 was used on SpeI digested DNA, and probe 2 was used on BglI digested DNA.