Attempts to test functionality of the -2232 C/G site using rhCRHH1 and H2 reporter constructs (−3458 to +974) generated inconsistent results because of the fact that they exhibited both extremely low levels of transfection efficiency and increased cell mortality, which we attributed to size of these constructs. This was true among experiments performed in a variety of cell lines (COS-7, IV-B, HT22, and JEG). GRE half-sites are known to interact across long distances 25, and there are several GREs and GRE half-sites in the proximal promoter of the CRH gene. Since we specifically wanted to test the function of the -2232 C/G polymorphism, located in a more distal GRE half-site, we designed double-stranded DNA cassettes (-2269 > -2200) containing either the -2232 C or -2232 G allele using oligonucleotides obtained from a commercial vendor (Operon, Huntsville, AL). These cassettes were digested with BglII and HindIII and ligated into the pDsRed MCS in a position 5′ to the rhCRH −660 to +123 insert (which contained the proximal promoter). Fidelity of resultant constructs was verified by sequencing.
