Sanyuan Ma

Advanced silk material spun by a transgenic silkworm promotes cell proliferation for biomedical application

Natural silk fiber spun by the silkworm Bombyx mori is widely used not only for textile materials, but also for biofunctional materials. In the present study, we genetically engineered an advanced silk material, named hSFSV, using a transgenic silkworm, in which the recombinant human acidic fibroblast growth factor (hFGF1) protein was specifically synthesized in the middle silk gland and secreted into the sericin layer to surround the silk fiber using our previously optimized sericin1 expression system. The content of the recombinant hFGF1 in the hSFSV silk was estimated to be approximate 0.07% of the cocoon shell weight. The mechanical properties of hSFSV raw silk fiber were enhanced slightly compared to those of the wild-type raw silk fiber, probably due to the presence of the recombinant of hFGF1 in the sericin layer. Remarkably, the hSFSV raw silk significantly stimulated the cell growth and proliferation of NIH/3T3 mouse embryonic fibroblast cells, suggesting that the mitogenic activity of recombinant hFGF1 was well maintained and functioned in the sericin layer of hSFSV raw silk. These results show that the genetically engineered raw silk hSFSV could be used directly as a fine biomedical material for mass application. In addition, the strategy whereby functional recombinant proteins are expressed in the sericin layer of silk might be used to create more genetically engineered silks with various biofunctions and applications.

Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains

Abstract  To overcome the disadvantages of current silkworm Bombyx mori transgenic technology, such as costly and time‐consuming to maintain non‐diapause transgenic silkworms, we report here on the development of treatments for the germline transformation of diapause silkworm strains. Our results showed that HCl treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains. By incubating developing mother eggs from Chinese lineage diapause silkworm strains at 15°C (15°C‐IME), we were able to prevent the diapause of their daughter eggs; a similar strategy (15°C‐IMES) for the germline transformation of the same strains was that the mother eggs were incubated at 15°C, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non‐diapause eggs. By combining temperature and light controls, the improved 15°C‐IMES strategy prevented diapause in daughter eggs, and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains. Although each of the strategies developed here has advantages and disadvantages, we suggest that the 15°C‐IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects. These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also highlight silkworm functional genomics research and its modeling.

Genetic marking of sex using a W chromosome-linked transgene.

Many species belonging to the order Lepidoptera are major pests in agriculture and arboriculture. The sterile insect technique (SIT) is an eco-friendly and highly efficient genetically targeted pest management approach. In many cases, it is preferable to release only sterile males in an SIT program, and efficient sexing strategies are crucial to the successful large-scale implementation of SIT. In the present study, we established 160 transgenic silkworm (Bombyx mori) lines to test the possibility of genetic sexing using a W chromosome-linked transgene, which is thought to be the best sexing strategy for lepidopteran species. One transgenic line with a female-specific expression pattern of reporter gene was obtained. The expression level of the W-linked transgene was comparable with autosomal insertions and was stable for 17 continuous generations. Molecular characterization showed this line contained a single copy of the reporter gene on the W chromosome, and the integration site was TTAG in contig W-BAC-522N19-C9. The feasibility of using a W chromosome-linked transgene demonstrated here and the possible improvements discussed will provide valuable information for other lepidopteran pests. The novel W chromosome-linked transgenic line established in this study will serve as an important resource for fundamental research with the silkworm B. mori.

Remobilizing deleted piggyBac vector post-integration for transgene stability in silkworm

Deletion of transposable elements post-genomic integration holds great promise for stability of the transgene in the host genome and has an essential role for the practical application of transgenic animals. In this study, a modified piggyBac vector that mediated deletion of the transposon sequence post-integration for transgene stability in the economically important silkworm Bombyx mori was constructed. The piggyBac vector architecture contains inversed terminal repeat sequences L1, L2 and R1, which can form L1/R1 and L2/R1 types of transposition cassettes. hsp70-PIG as the piggyBac transposase expression cassette for initial transposition, further remobilization and transgene stabilization test was transiently expressed in a helper vector or integrated into the modified vector to produce a transgenic silkworm. Shortening L2 increased the transformation frequency of L1/R1 into the silkworm genome compared to L2/R1. After the integration of L1/R1 into the genome, the remobilization of L2/R1 impaired the transposon structure and the resulting transgene linked with an impaired transposon was stable in the genome even in the presence of exogenously introduced transposase, whereas those flanked by the intact transposon were highly mobile in the genome. Our results demonstrated the feasibility of post-integration deletion of transposable elements to guarantee true transgene stabilization in silkworm. We suggest that the modified vector will be a useful resource for studies of transgenic silkworms and other piggyBac-transformed organisms.

Bacillus bombysepticus α-Toxin Binding to G Protein-Coupled Receptor Kinase 2 Regulates cAMP/PKA Signaling Pathway to Induce Host Death

Bacterial pathogens and their toxins target host receptors, leading to aberrant behavior or host death by changing signaling events through subversion of host intracellular cAMP level. This is an efficient and widespread mechanism of microbial pathogenesis. Previous studies describe toxins that increase cAMP in host cells, resulting in death through G protein-coupled receptor (GPCR) signaling pathways by influencing adenylyl cyclase or G protein activity. G protein-coupled receptor kinase 2 (GRK2) has a central role in regulation of GPCR desensitization. However, little information is available about the pathogenic mechanisms of toxins associated with GRK2. Here, we reported a new bacterial toxin-Bacillus bombysepticus (Bb) α-toxin that was lethal to host. We showed that Bb α-toxin interacted with BmGRK2. The data demonstrated that Bb α-toxin directly bound to BmGRK2 to promote death by affecting GPCR signaling pathways. This mechanism involved stimulation of Gαs, increase level of cAMP and activation of protein kinase A (PKA). Activated cAMP/PKA signal transduction altered downstream effectors that affected homeostasis and fundamental biological processes, disturbing the structural and functional integrity of cells, resulting in death. Preventing cAMP/PKA signaling transduction by inhibitions (NF449 or H-89) substantially reduced the pathogenicity of Bb α-toxin. The discovery of a toxin-induced host death specifically linked to GRK2 mediated signaling pathway suggested a new model for bacterial toxin action. Characterization of host genes whose expression and function are regulated by Bb α-toxin and GRK2 will offer a deeper understanding of the pathogenesis of infectious diseases caused by pathogens that elevate cAMP.