Construction of a binary transgenic gene expression system for recombinant protein production in the middle silk gland of the silkworm Bombyx mori

Abstract

To construct an efficient system for the production of recombinant proteins in silkworm (Bombyx mori), we investigated the promoter activity of the silkworm sericin 1, 2, and 3 genes (Ser1, Ser2, and Ser3) using a GAL4/UAS binary gene expression system in transgenic silkworm. The promoter activity of the upstream region of Ser1 was strong, yielding high expression of an enhanced green fluorescent protein (EGFP) transgene in the middle and posterior regions of the middle silk gland (MSG) after day 2 of the fifth instar. The Ser3 upstream region exhibited moderate promoter activity in the anterior MSG, but the Ser2 upstream region did not exhibit any promoter activity. Since the strongest promoter activity was observed for Ser1, we devised a system for the production of recombinant proteins using a GAL4-Ser1 promoter construct (Ser1-GAL4). Transgenic silkworms harboring both the Ser1-GAL4 construct and the previously reported upstream activating sequence (UAS)-EGFP construct, which contains the TATA box region of the Drosophila hsp70 gene, yielded approximately 100 microg EGFP per larva. When we then analyzed the TATA box region, signal peptide, and intron sequences for their effects on production from the UAS-EGFP construct, we found that the optimization of these sequences effectively increased production to an average of 500 microg EGFP protein per transgenic larva. We conclude that this binary system is a useful tool for the mass production of recombinant proteins of biomedical and pharmaceutical interest in silkworm.

CD36 homolog divergence is responsible for the selectivity of carotenoid species migration to the silk gland of the silkworm Bombyx mori

“…6D ). This pattern was concordant with the property of the Ser1-GAL4 driver as monitored using the UAS-EGFP gene ( 29 ). On the other hand, the pigmentation pattern in larvae bearing the C allele commences from the middle part of the gland but spreads into the posterior region only minimally ( Fig.…”

“…For the effector strains, the effector construct and the helper plasmid pHA3PIG ( 28 ) were injected into preblastoderm embryos of the strain w1-pnd-925, a nondiapausing strain with the phenotype of yellow hemolymph and white cocoons ( 17 ). The existence of the transgene was identifi ed by the fl uorescence in the eye, EGFP for UAS-SCRB15 and DsRed2 for Ser1-GAL4 ( 29 ). Individuals exhibiting colorless hemolymph in the larval stage, which had colorless silk glands and produced white cocoons, were not used to examine the color phenotype or carotenoid composition of the middle silk gland and cocoons.…”

Recent progress in development of transgenic silkworms overexpressing recombinant human proteins with therapeutic potential in silk glands

“…A cloned open reading frame (ORF) fragment of CTSA was inserted downstream of the upstream activation sequence (UAS) of the piggyBac vector pBac[UAS-3xP3-EGFP] plasmid (21). The MSG-specific sericin I gene promoter (Ser1p) and its 3'-UTR region, including the BlnI site, was amplified from the genomic DNA of B. mori with PCR to construct the plasmid pBac[Ser1p-BlnI-UTR, 3xP3-DsRed2] (22), and this plasmid was inserted into regions including the Nhe I and Bgl II sites of pBacMCS (23,24). To construct the plasmid pBac[Ser1p-Gal4VP16, 3xP3-DsRed2], a Gal4VP16 NheI fragment (22) was inserted into the BlnI site of the plasmid pBac[Ser1-BlnI-UTR, 3xP3-DsRed2].…”

A CD36-related Transmembrane Protein Is Coordinated with an Intracellular Lipid-binding Protein in Selective Carotenoid Transport for Cocoon Coloration

“…For the effector strains, the effector construct and the helper plasmid, pHA3PIG (29), were injected into preblastoderm embryos of the w1-pnd-925 strain at a concentration of 0.2 mg/ml. After sib selection based on the presence of EGFP fluorescence in the eye by the 3xP3-EGFP gene, G1 male moths of a UAS-Cameo2 (UAS) line with the phenotype of yellow hemolymph and white cocoons were crossed with females of the Ser1-GAL4 (GAL4) line with the phenotype of colorless hemolymph and white cocoons, which drives target gene expression in the middle silk gland and has a marker fluorescence in the eye by the 3xP3-DsRed gene (31). Because the transgene was supposed to be homozygous in the GAL4 line, the progeny of the cross between the UAS line and GAL4 line showed two different marker phenotypes of eye color: both DsRed-and EGFP-positive, GAL4/ UAS line (Ser1-GAL4(ϩ), UAS-Cameo2(ϩ)); and only DsRedpositive, GAL4 line (Ser1-GAL4(ϩ), UAS-Cameo2(Ϫ)).…”

“…7A) and then the effector UAS-Cameo2 (UAS) lines were generated by germ line transformation. Male moths of a UAS line were crossed with females of the Ser1-GAL4 (GAL4) line that drives target gene expression in the middle silk gland (31). The restoration of pigmentation in the middle silk gland was observed in the GAL4/UAS line (Fig.…”