Figure 6. Analysis of regulatory elements in the IGR responsible for hypothalamic VP gene expression. A, The VP constructs used in this IGR analysis were based on the 288VPIII.EGFP.IGR2.1 construct in which the 2.1 kb IGR was divided into five segments (A-E). Each segment was linked to 288VPIII.EGFP. ΔIGR construct contained no IGR sequence (Fig. 5), thereby producing the other constructs with IGR inserts (A-E) of length 178, 376, 298, 446, or 834 bp, respectively (see Materials and Methods). B, Results from the biolistic transfection of rat hypothalamic cultures using the VP constructs shown in A. Cultures were assayed by IHC of EGFP using fluorescent microscopy, and EGFP-expressing neurons were counted per filter. Data are expressed as averages ± SEMs. Note that the enhancer activity in the 2.1 kb IGR appears to be primarily located in the 178 bp segment (A). 2.1, 288VPIII.EGFP.IGR2.1; A, 288VPIII.EGFP.IGR178; B, 288VPIII.EGFP.IGR376; C, 288VPIII.EGFP.IGR298; D, 288VPIII.EGFP.IGR446; E, 288VPIII.EGFP.IGR834.