embryo transfer. Recipientscarryingapalpable corpus luteum ([0.8 cm) at day 7 after estrus detection were selected for embryo transfer. Morulae or blastocysts were nonsurgically transferred to the uterine horn ipsilateral to the corpus luteum (two embryos per recipient). Ultrasonic testing was performed approximately 50 days after embryo transfer.
Mfat1 gene insertion analysis
Genomic DNA was extracted from tissue or blood of the cloned calves named fat1 1-20 using Genomic DNA extraction Kit. Primers were designed to analyze the insertion of the mfat1 gene (P1, 50-GGACCTGGT GAAGAGCATCCG-30; P2, 5 0-GCGTCGCAGAAGC CAAAC-30, corresponding to 210–230 and 630– 647 bp of C. elegans fat1 gene, respectively).
Expression analysis of mfat1 gene
Total RNA from four tissues (muscle, liver, lung, and kidney)waspurifiedusingatotalRNAextractionkit.The reverse-transcriptionprocedurewas42 Cfor60 minand 70 C for 5 min. RT-PCR was performed using primers P1 and P2 and the procedure was described above. The 297 bp beta-actin band was amplified as a control for the quality of RT-PCR (P3, 50-CCAACCGTGAGAAGAT GAC-30; P4, 5 0-TGAGTCACGGACGATTTC-30).