Generation of hybrid transgenic silkworms that express Bombyx mori prolyl-hydroxylase alpha-subunits and human collagens in posterior silk glands: Production of cocoons that contained collagens with hydroxylated proline residues.

Prolyl 4-hydroxylase (P4H) is a heterotetramer enzyme consisting of alpha-subunits (P4Halpha) and beta-subunits (P4Hbeta), and is required for collagen biosynthesis. Previously, we generated transgenic silkworms that produced human type III collagen fragments (mini-collagens) in the posterior silk gland (PSG). However, prolyl 4-hydroxylation did not occur on the mini-collagens, because in spite of an abundant expression of P4Hbeta in PSGs, P4Halpha expression was quite low there, thus resulting in an insufficient activity of P4H. In this study we aimed at generating hybrid transgenic silkworms whose PSGs are capable of producing mini-collagens and enough P4H for their prolyl 4-hydroxylation. Isolated PSGs were bombarded with fibroin L-chain gene promoter-driven vectors containing Bombyx mori P4Halpha (BmP4Halpha) cDNAs and were transplanted into the hemolymphatic cavity. The P4H activity in the PSG cells significantly increased, indicating that the expressed BmP4Halpha formed active tetramers with endogenous BmP4Hbeta. Using germ-line transgenesis technology, silkworms were generated that synthesized BmP4Halpha in PSG cells. The P4H activity in the transgenic silkworms was 130-fold higher than that of wild-type counterparts. Finally, we generated hybrid transgenic silkworms that expressed cDNAs of both BmP4Halpha and mini-collagen in PSG cells. They spun cocoons that contained mini-collagens whose appropriate proline residues had been adequately hydroxylated.