The pPL657 recombinant DNA construct was designed to disrupt the endogenous glycoprotein galactosyltransferase alpha-1,3 (ggta1) gene. The construct consisted of ggta1 sequences flanking an Escherichia coli neomycin phosphotransferase II (nptII) cassette. The ggta1 sequences are homologous to intron 8 and exon 9 of the endogenous ggta1 gene. Homologous recombination inserted the nptII cassette in the 5' end of the ggta1 exon 9. The nptII cassette contains a Bos taurus bovine growth hormone terminator, preventing the transcription of exon 9, which contains the catalytic domains of the GGTA1 enzyme.

Expression of nptII relies on the Encephalomyocarditis virus internal ribosome binding site. The internal ribosome binding site allows for initiation of translation in a cap-independent manner. Thus, translation can occur from both the 5' end of the GGTA1 transcript and this nptII cassette.

The knockout cassette (internal ribosome binding site - nptII - bovine growth factor terminator) is roughly 1.8 kb in size.

Long range PCR and Sanger sequencing indicated that the insert occurred in intron 8 and exon 9 of the ggta1 gene. No random (off-target) insertions or vector backbone fragments were detected. Sequencing of later generations from the founder pig indicated that the sequences were substantially similar. The lack of detectable alpha-gal continued into later generations (F11 generation was analyzed).