A 2,346-base pair SstI/HindIII fragment of the rat β4 subunit gene was treated with Klenow fragment of DNA polymerase I to generate blunt ends. This fragment spans nucleotides −2208 to +137 relative to the β4 transcription initiation site (Hu et al., 1994). The blunt-ended SstI/HindIII fragment was subcloned into the β-gal expression vector, pSG-MAR, which had been digested with SmaI, to generate the construct pSGB4SH (Fig. 1B). pSG-MAR contains the β-gal coding sequence, a nuclear localization signal (NLS) as well as the 5′ matrix attachment region (MAR) of the chick lysozyme gene (Fuchs et al., 2002). The NLS was included to facilitate comparison between β-gal activity with β4 RNA expression and to reduce background staining (Mercer et al., 1991). The MAR was included to insulate the transgene from insertion positional effects (Phi-Van and Stratling, 1996). The construct was verified by nucleotide sequencing (Nucleic Acid Facility, University of Massachusetts Medical School).
