Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential. Significance Specific gene functions have been successfully suppressed by gene silencing or editing in many organisms. However, genetic manipulation to suppress the function of a target tissue has not been achieved using cytotoxin genes. We established transgenic silkworms with posterior silk glands (PSGs) that express the enzymatic domain of the cytotoxin pierisin-1A (P1A). The larvae with the modified PSGs produced the sericin cocoons with potential utilities in tissue engineering. The targeted P1A expression was found to cause site-specific repression of certain protein synthesis that appeared to have no impact on the developmental stages of individuals. Thus, the new approach through targeted P1A expression could be applicable to the development of biologically useful model organisms with tissue-specific dysfunction.

Silk fibroin exhibits high biocompatibility and biodegradability, making it a versatile biomaterial for medical applications. However, contaminated silkworm-derived substances in remnant sericin from the filature and degumming process can result in undesired immune reactions and silk allergy, limiting the widespread use of fibroin. Here, we established transgenic silkworms with modified middle silk glands, in which sericin expression was repressed by the ectopic expression of cabbage butterfly-derived cytotoxin pierisin-1A, to produce cocoons composed solely of fibroin. Intact, nondegraded fibroin can be prepared from the transgenic cocoons without the need for sericin removal by the filature and degumming steps that cause fibroin degradation. A wide-angle X-ray diffraction analysis revealed low crystallinity in the transgenic cocoons. However, nondegraded fibroin obtained from transgenic cocoons enabled the formation of fibroin sponges with varying densities by using 1–5% (v/v) alcohol. The effective chondrogenic differentiation of ATDC5 cells was induced following their cultivation on substrates coated with intact fibroin. Our results showed that intact, allergen-free fibroin can be obtained from transgenic cocoons without the need for sericin removal, providing a method to produce fibroin-based materials with high biocompatibility for biomedical uses.

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.

Growth factors, including fibroblast growth factor-7 (FGF-7), are a group of proteins that stimulate various cellular processes and are often used with carriers to prevent the rapid loss of their activities. Sericin with great biocompatibility has been investigated as a proteinaceous carrier to enhance the stability of incorporated proteins. The difficulties in obtaining intact sericin from silkworm cocoons and the handling of growth factors with poor stability necessitate an efficient technique to incorporate the protein into a sericin-based biomaterial. Here, we report the generation of a transgenic silkworm line simultaneously expressing and incorporating FGF-7 into cocoon shells containing almost exclusively sericin. Growth-factor-functionalized sericin cocoon shells requiring simple lyophilization and pulverization processes were successfully used to induce the proliferation and migration of keratinocytes. Moreover, FGF-7 incorporated into sericin-cocoon powder exhibited remarkable stability, with more than 70% of bioactivity being retained after being stored as a suspension at 25 °C for 3 months. Transgenic sericin-cocoon powder was used to continuously supply biologically active FGF-7 to generate a three-dimensionally cultured keratinocyte model in vitro. The outcomes of this study propound a feasible approach to producing cytokine-functionalized sericin materials that are ready to use for cell cultivation.

Silk gut processed from the silk glands of the silkworm could be an ideal biodegradable carrier for cell growth factors. We previously demonstrated that polyhedra, microcrystals of Cypovirus 1 polyhedrin, can serve as versatile carrier proteins. Here, we report the generation of a transgenic silkworm that expresses polyhedrin together with human basic fibroblast growth factor (FGF-2) in its posterior silk glands to utilize silk gut as a proteinaceous carrier to protect and slowly release active cell growth factors. In the posterior silk glands, polyhedrin formed polyhedral microcrystals and FGF-2 became encapsulated within the polyhedra due to a polyhedron-immobilization signal. Silk gut powder prepared from posterior silk glands containing polyhedron-encapsulated FGF-2 stimulated the phosphorylation of p44/p42 MAP kinase and induced the proliferation of serum-starved NIH3T3 cells by releasing bioactive FGF-2. Even after a one-week incubation at 25 °C, significantly higher biological activity of FGF-2 was observed for silk gut powder incorporating polyhedron-encapsulated FGF-2 relative to silk gut powder with non-encapsulated FGF-2. Our results demonstrate that posterior silk glands incorporating polyhedron-encapsulated FGF-2 are applicable to the preparation of biodegradable silk gut, which can protect and release FGF-2 that is produced in a virus- and serum-free expression system with significant application potential.

Sesame seeds are abundant in sesamin, which exerts health-promoting effects such as extending the lifespan of adult Drosophila and suppressing oxidative stress by activating the Nrf2 transcription factor. Here, we investigated whether sesamin activated Nrf2 in larval tissues and induced the expression of Nrf2 target genes. In the sesamin-fed larvae, Nrf2 was activated in the central nervous system (CNS), gut, and salivary glands. The ectopic expression of Keap1 in glial cells inhibited sesamin-induced Nrf2 activation in the whole CNS more than in the neurons, indicating that sesamin activates Nrf2 in glia efficiently. We labeled the astrocytes as well as cortex and surface glia with fluorescence to identify the glial cell types in which Nrf2 was activated; we observed their activation in both cell types. These data suggest that sesamin may stimulate the expression of antioxidative genes in glial cells. Among the 17 candidate Nrf2 targets, the mRNA levels of Cyp6a2 and Cyp6g1 in cytochrome P450 were elevated in the CNS, gut, and salivary glands of the sesamin-fed larvae. However, this elevation did not lead to resistance against imidacloprid, which is detoxified by these enzymes. Our results suggest that sesamin may exert similar health-promoting effects on the human CNS and digestive tissues.