RESUMEN
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.
Asunto(s)
ADP Ribosa Transferasas , Animales Modificados Genéticamente , Bombyx , Citotoxinas , Glándulas Exocrinas/metabolismo , Hidrogeles/farmacología , Proteínas de Insectos , Células Madre Embrionarias de Ratones/metabolismo , Sericinas , ADP Ribosa Transferasas/biosíntesis , ADP Ribosa Transferasas/genética , ADP Ribosa Transferasas/farmacología , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Bombyx/genética , Bombyx/metabolismo , Citocinas/biosíntesis , Citotoxinas/biosíntesis , Citotoxinas/genética , Citotoxinas/farmacología , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/genética , Proteínas de Insectos/farmacología , Ratones , Células Madre Embrionarias de Ratones/citología , Sericinas/biosíntesis , Sericinas/genética , Sericinas/farmacologíaRESUMEN
Non-thermal atmospheric-pressure plasma (NTAPP) has been widely studied for clinical applications, e.g., disinfection, wound healing, cancer therapy, hemostasis, and bone regeneration. It is being revealed that the physical and chemical actions of plasma have enabled these clinical applications. Based on our previous report regarding plasma-stimulated bone regeneration, this study focused on Achilles tendon repair by NTAPP. This is the first study to reveal that exposure to NTAPP can accelerate Achilles tendon repair using a well-established Achilles tendon injury rat model. Histological evaluation using the Stoll's and histological scores showed a significant improvement at 2 and 4 weeks, with type I collagen content being substantial at the early time point of 2 weeks post-surgery. Notably, the replacement of type III collagen with type I collagen occurred more frequently in the plasma-treated groups at the early stage of repair. Tensile strength test results showed that the maximum breaking strength in the plasma-treated group at two weeks was significantly higher than that in the untreated group. Overall, our results indicate that a single event of NTAPP treatment during the surgery can contribute to an early recovery of an injured tendon.