Genomic DNA was obtained from tail biopsies. Tail tissue was lysed in buffer containing 100 mM tris-HCl (pH 8.5), 5 mM EDTA, 0.2% SDS, 200 mM NaCl, and proteinase K (100 μg/ml) (Roche) overnight at 55°C. Genomic DNA was extracted by phenol-chloroform and chloroform, followed by precipitation with 2.5 volumes of isopropanol and washing with 70% ethanol. The DNA pellet was dissolved in TE buffer [10 mM tris (pH 7.9) and 0.2 mM EDTA]. Positively targeted F1 animals were analyzed using Southern blot hybridization. Approximately 10 to 20 μg of genomic DNA was digested with the corresponding restriction endonuclease, fractionated on 0.8% agarose gels, and transferred to GeneScreen nylon membranes (NEN DuPont). The membranes were hybridized with 32P-labeled specific DNA probes (table S2). DNA labeling was performed using a random prime DNA labeling kit (Roche) and [α-32P] deoxycytidine-5′ triphosphate (PerkinElmer). Membranes were washed with 0.5× saline sodium phosphate EDTA (SSPE) buffer [1× saline sodium phosphate EDTA buffer is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA (pH 7.7)] and 0.5% SDS at 65°C and exposed to MS film (Kodak) at −80°C.

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