Mutation of gene-proximal regulatory elements disrupts human ɛ-, γ-, and β-globin expression in yeast artificial chromosome transgenic mice

The YAC transgene copy number in the lines chosen for final expression analysis was determined using a YAC end assay, employing a probe that would detect a common 1.6-kbp band as well as other characteristic fragments in each digest. For example, a head-to-head tandem integration was predicted from the map of pYAC4 to yield an additional fragment 4.52 kbp in size (Fig. 2A, lanes 3 and 6), whereas a head-to-tail tandem integration would result in the generation of a fragment of 5.36 kbp (Fig. 2A, lane 5); if the YAC end was embedded in mouse chromosomal DNA (as either a single copy transgene or as the flanking copy of a multi-YAC integration), the size of the fragment would be dependent on the next random PstI site encountered in the mouse genome. From these data, combined with the data obtained from standard Southern blots and comparison of internal fragment intensities, we estimate that the three ɛ-silencer deletion lines contain three, two, and two transgene copies (Fig. 2A, lanes 1–3), respectively, whereas the two β-globin enhancer deletion lines both harbor four tandemly integrated YAC copies (Fig. 2A, lanes 5 and 6). Furthermore, the analysis shows that, as previously stated, the wild-type YAC transgenic line HS4321b (26) is single copy (Fig. 2A, lane 4).