Human epidermal growth factor-functionalized cocoon silk with improved cell proliferation activity for the fabrication of wound dressings

Human epidermal growth factor (hEGF) is a key factor involved in wound healing owing to its powerful ability to stimulate cell proliferation. In this study, we used piggyBac transposon technology to produce transgenic silkworms expressing the hEGF protein fused to truncated heavy chain (FibH-hEGF). The FibH–hEGF fusion protein was successfully expressed and secreted into silkworm cocoons. Compared to wild-type silk, the transgenic silkworm silk had the similar morphology about silks fiber surface and cocoon nets, while the secondary structure between the transgenic silk and wild-type silk was different. Most importantly, transgenic silkworm cocoon silk powder extract significantly increased human fibroblast FIB cell proliferation for a long duration with no apparent cytotoxicity. Our study provides a promising method for obtaining cost-effective and functional biomaterials for the fabrication of wound dressings.

Toxicological evaluation of transgenic silkworms

Safety of transgenic silkworms must be evaluated before their commercial application. We assessed subacute toxicity using a 28-day feeding study in rats. Eighty rats were evenly allocated into four groups, with each group containing 10 male and 10 female rats. Rats of three groups were fed dried transgenic silkworm H19.9A pupae with overexpressed endogenous Bmhsp19.9, transgenic silkworm A4SOR pupae with overexpressed exogenous SOR, or normal silkworm pupae at a dose of 3.0 g/kg/day, respectively. The fourth group served as a normal feeding control. The body weight, feed consumption, hematology response variables, serum biochemical parameters, organ weights, gross necropsy, and histopathologic of animals were evaluated. No mortality, adverse effects, or major differences in the evaluated parameters were observed in the groups fed transgenic pupae in comparison with the control, suggesting that transgenic silkworms are toxicologically equivalent to normal silkworms and are safe for consumption in rats.

Structural insight and stability of TNFR-Fc fusion protein (Etanercept) produced by using transgenic silkworms

Therapeutic proteins expressed using transgenic animals have been of great interest for several years. Especially, transgenic silkworm has been studied intensively because of its ease in handling, low-cost, high-yield and unique glycosylation patterns. However, the physicochemical property of the therapeutic protein expressed in transgenic silkworm remains elusive. Here, we constructed an expression system for the TNFR-Fc fusion protein (Etanercept) using transgenic silkworm. The TNFR-Fc fusion protein was employed to N-glycan analysis, which revealed an increased amount of afucosylated protein. Evidence from surface plasmon resonance analysis showed that the TNFR-Fc fusion protein exhibit increased binding affinity for Fcγ receptor IIIa and FcRn compared to the commercial Etanercept, emphasizing the profit of expression system using transgenic silkworm. We have further discussed the comparison of higher order structure, thermal stability and aggregation of the TNFR-Fc fusion protein.

Spliceosomal Protein Gene BmSPX Regulates Reproductive Organ Development in Bombyx mori

Sex determination and differentiation are nearly universal to all eukaryotic organisms, encompassing diverse systems and mechanisms. Here, we identified a spliceosomal protein gene BmSPX involved in sex determination of the lepidopeteran insect, Bombyx mori. In a transgenic silkworm line that overexpressed the BmSPX gene, transgenic silkworm males exhibited differences in their external genitalia compared to wild-type males, but normal internal genitalia. Additionally, transgenic silkworm females exhibited a developmental disorder of the reproductive organs. Upregulation of BmSPX significantly increased the expression levels of sex-determining genes (BmMasc and BmIMP) and reduced the female-type splice isoform of Bmdsx, which is a key switch gene downstream of the sex-determination pathway. Additionally, co-immunoprecipitation assays confirmed an interaction between the BmSPX protein and BmPSI, an upstream regulatory factor of Bmdsx. Quantitative real-time PCR showed that BmSPX over-expression upregulated the expression of the Hox gene abdominal-B (Adb-B), which is required for specification of the posterior abdomen, external genitalia, and gonads of insects, as well as the genes in the Receptor Tyrosine Kinase (RTK) signaling pathway. In conclusion, our study suggested the involvement of BmSPX, identified as a novel regulatory factor, in the sex-determination pathway and regulation of reproductive organ development in silkworms.

CRISPR/Cpf1 mediated genome editing enhances Bombyx mori resistance to BmNPV

CRISPR/Cas12a (Cpf1) is a single RNA-guided endonuclease that provides new opportunities for targeted genome engineering through the CRISPR/Cas9 system. Only AsCpf1 have been developed for insect genome editing, and the novel Cas12a orthologs nucleases and editing efficiency require more study in insect. We compared three Cas12a orthologs nucleases, AsCpf1, FnCpf1, and LbCpf1, for their editing efficiencies and antiviral abilities in vitro. The three Cpf1 efficiently edited the BmNPV genome and inhibited BmNPV replication in BmN-SWU1 cells. The antiviral ability of the FnCpf1 system was more efficient than the SpCas9 system after infection by BmNPV. We created FnCpf1×gIE1 and SpCas9×sgIE1 transgenic hybrid lines and evaluated the gene editing efficiency of different systems at the same target site. We improved the antiviral ability using the FnCpf1 system in transgenic silkworm. This study demonstrated use of the CRISPR/Cpf1 system to achieve high editing efficiencies in the silkworm, and illustrates the use of this technology for increasing disease resistance.Author SummaryGenome editing is a powerful tool that has been widely used in gene function, gene therapy, pest control, and disease-resistant engineering in most parts of pathogens research. Since the establishment of CRISPR/Cas9, powerful strategies for antiviral therapy of transgenic silkworm have emerged. Nevertheless, there is still room to expand the scope of genome editing tool for further application to improve antiviral research. Here, we demonstrate that three Cpf1 endonuclease can be used efficiency editing BmNPV genome in vitro and in vivo for the first time. More importantly, this Cpf1 system could improve the resistance of transgenic silkworms to BmNPV compare with Cas9 system, and no significant cocoons difference was observed between transgenic lines infected with BmNPV and control. These broaden the range of application of CRISPR for novel genome editing methods in silkworm and also enable sheds light on antiviral therapy.

Direct Recovery of the Rare Earth Elements Using a Silk Displaying a Metal-Recognizing Peptide

Rare earth elements (RE) are indispensable metallic resources in the production of advanced materials; hence, a cost- and energy-effective recovery process is required to meet the rapidly increasing RE demand. Here, we propose an artificial RE recovery approach that uses a functional silk displaying a RE-recognizing peptide. Using the piggyBac system, we constructed a transgenic silkworm in which one or two copies of the gene coding for the RE-recognizing peptide (Lamp1) was fused with that of the fibroin L (FibL) protein. The purified FibL-Lamp1 fusion protein from the transgenic silkworm was able to recognize dysprosium (Dy3+), a RE, under physiological conditions. This method can also be used with silk from which sericin has been removed. Furthermore, the Dy-recovery ability of this silk was significantly improved by crushing the silk. Our simple approach is expected to facilitate the direct recovery of RE from an actual mixed solution of metal ions, such as seawater and industrial wastewater, under mild conditions without additional energy input.