Poly A⁺ RNA was prepared from group A porcine submaxillary glands and used for RT-PCR of a putative porcine A transferase complementary DNA using 2 primers: FY-530 (5′-CTCCAGGCACCTGGCTTG) and FY-531 (5′-CCCTCCTCCTGTTCGTCG). The sequences in these primers corresponded to the sequences in the 5′ UTR and the 3′ UTR regions of the complementary DNA. The PCR product was cloned into pT-Adv vector by using the T-A cloning method and subsequently transferred into the pSG-5 eukaryotic expression vector. DNA from the pPigA expression constructs was used for the Lipofectamine (Gibco, Grand Island, NY)-mediated DNA transfection assays. The human uterus–derived adenocarcinoma cell line HeLa was the recipient, as described previously.10 11 Human A and B transferase expression constructs pAAAA and pBBBB were used as positive controls, and nonfunctional pA(arg) construct was used as a negative control. Two days after transfection, some floating cells were plated into a 96-well plate. The next day, cells were fixed and immunostained with anti-A and anti-B murine monoclonal antibody mixtures, avidin-biotin complex reagents, and 3,3′-diaminobenzidine substrates. The cells from the original plates were harvested and lysed, and A and B transferase activity was determined by measuring the transfer of ¹⁴C from ¹⁴C-UDP-N-acetyl-D-galactosamine and ¹⁴C-UDP-galactose, respectively, to the acceptor substrate 2′-fucosyllactose. The reactions without 2′-fucosyllactose were used as negative controls. A summary of the activity of the constructs is shown in the last column.

RT-PCR indicates reverse transcriptase–polymerase chain reaction; UTR, untranslated region; UDP, uridine diphosphate.