Genomic DNA from animals confirmed to be homozygous for the rhCRH-H1 haplotype was extracted for PCR-based cloning. Primers were modified from those previously reported for huCRH reporter constructs 24. The forward primer was 5′ GCG GAA TTC GGC TCA TAA CTC CTT TAT GTG CTT GC 3′ (containing an EcoRI site) and the reverse primer was 5′ AAA GGA TCC GAG GGA CGT CTC CGG GGC 3′ (containing a BamHI site). These primers produced a 783-bp amplification product (from rhCRH −660 to +123) in which none of the rhCRH-H2 co-segregating loci were present. Reaction mixtures (50μl) contained 100 ng DNA, 0.1 mM dNTPs, 0.5 μM of each primer, 2.5 U PfuUltra High Fidelity DNA Polymerase and PfuUltra Buffer (Stratagene, La Jolla, CA). Amplifications were performed using a Perkin Elmer thermocycler (9700) with one cycle at 95∞C, 30 cycles of 95∞C/30 sec, 61.5∞C/30 sec, 72∞C/5 min and a final 10-minute extension at 72∞C. Following cleavage using EcoR1 and BamHI, PCR product was separated by electrophoresis and isolated using a QIAquick gel extraction kit (Qiagen, Valencia, CA). Cleaved products were then ligated into EcoRI/BamHI digested pDsRed-2.1 (BD Biosciences, Mountainview, CA) using standard molecular cloning techniques.
