Figure 3
Analysis of the final construct. Two CreERT2-Neo positive clones were analyzed by restriction with Mss I (lanes 1 and 2). This resulted in the 42 kb insert (upper band) and the 2.8 kb long backbone (lower band). The restriction of the two constructs with Hin dIII resulted in the expected restriction pattern of 17.9 kb 7.9 kb, 4.5 kb, 3.6 kb, 3.1 kb, 2.5 kb, 2.2 kb, 1.9 kb and 1 kb (lanes 3 and 4). Uncut clones are shown in lanes 5 and 6. In lane 7, the PCR generated targeting cassette of 2.9 kb is shown, which has almost the same size as the 2.8 kb released backbone after restriction with Mss I. The restriction of the CreERT2-Neo targeted BAC RP23-328P3 with Hin dIII resulted in fragments ranging from 13 bp to 14.9 kb (lane 8). The correct restriction pattern for both Mss I and Hin dIII demonstrated successful targeting and subcloning of the final construct.