Figure 2. Demonstration of transgenesis in cloned minipigs produced by HMC.

(A) Pictures of cloned Bama-minipigs at the age of five and 60 days. Curve indicates mean weight increase over the first 60 days of the 21 transgenic (hCry1^AP) and five non-transgenic (NT) animals (#321-1^wt, #321-2^wt, #321-3^wt, #321-5^wt, and #321-6^wt). (B) PCR and RT-PCR analysis on gDNA and total RNA, respectively, isolated from tail clips of the 23 cloned minipigs born from three recipient sows (#175, #377 and #208) as well as from one non-transgenic (NT) control. The PCR analysis employing hCry1 specific primers revealed genomic integration of the transgenic cassette in all the cloned animals (upper panel). RT-PCR analysis using exon-exon primers for hCry1 and porcine GAPDH showed robust expression of hCry1^AP in the transgenic animals with no detectable band in the lane corresponding to the NT control (lower two panels); PC, plasmid control; M, 100 bp marker. (C) Quantitative RT-PCR performed on cDNA from 14 of the 23 transgenic animals and a NT control (#321-1^wt). Total mRNA extracted from tail clips was reverse transcribed and used for quantification of hCry1 normalized to endogenous ACTB. The expression values are relative to the NT control. The experiment is performed in triplicate and data are presented as mean values ± standard deviation.