To determine whether the 2.3-kilobase pair β4 promoter region functions in vivo and if so, in a cell-type-specific manner reflecting endogenous β4 mRNA expression, the β4/β-gal transgene was excised from the pSGB4SH backbone and used to generate several founder lines of mice (see Experimental Procedures for details). Nine founder lines were obtained. Of these, 1 died unexpectedly, 1 showed no β-gal staining, 2 did not breed well and 1 showed extremely intense β-gal staining throughout the body and was not pursued. The remaining 4 lines (lines 30, 39, 54 and 447) were analyzed for β-gal staining at ED18.5 and PD30 as described below. Using a quantitative PCR approach (Yuan et al., 2007), transgene copy number was determined for each line and revealed that line 30 has approximately 7 copies, line 39 has 26 copies, line 54 has 12 copies and line 447 has 10 copies (see Experimental Procedures for details).
