Sericultural industry development professionals are interested in high-strength silk exceeding strength of 3–4 gf/d as a new source of added value of raw silk. Silkworm races possessed by National Agriculture and Food Research Organization include Shokei, with 6 gf/d of strength. Both Yoshi N and J124 have strength exceeding 5 gf/d. Using these races and the practical breeding stocks and materials, J149 and C515, we developed high-strength silkworms. By double crossing with J124×YoshiN and J149×J124, we repeated selection on the strength and passage from this crossing generation. A new Japanese strain called MN502 was fixed. In the Chinese strain, MC502 was fixed from a three-way hybrid of C515×208AB and Shokei. Comparing breeding results and reeling results of MN502×MC502 with those of hybrids, Hakurei®, Ariake and J137×C146, the cocoon weight and the cocoon shell weight were the lowest. Furthermore, the values of the cocoon filament length, the raw silk percentage and the amount of cocoon filament correlated with those of cocoon and cocoon-shell weight were also low. However, the silk thread strength showed its highest value at 4.99 gf/d. Its breeding was continued for strength. Therefore, it showed the value of the strength, MN502: 4.86, MC502: 5.25 and the hybrid: 5.31 gf/d in recent years. This genetic effect was superdominant, suggesting an effect of MC502.
Silk has excellent original properties. It is garnering attention not only as a material for clothing but also as an industrial material. Development of raw silk with even greater high strength and strain is anticipated in both cases. Raw silk made from the cocoon of MC502, a high-strength silk-thread producing Chinese silkworm strain, has greater than 5 g/d strength, although the yields of the cocoon and raw silk were lower than those of commercial silkworm strains. Therefore, MC502 was crossbred to five characteristic silkworm strains. Effects on cocoon thread character and raw silk yield were evaluated to improve the cocoon and raw silk yields necessary for the practical use of MC502. All F1-hybrid MC502 showed a significant increase of cocoon and cocoon shell weight. Furthermore, single cocoon reeling testing revealed that the cocoon filament length and size curve were changed relating to the crossbred silkworm strains. In addition, crossbreeding affected the reeling test score. The score was improved in most cases. Especially, a significant increase of raw silk percentage was observed when MC502 was crossbred with J137, a Japanese silkworm strain of standard character. All F1-hybrid MC502 maintained the high-strength character of MC502. The strengths of their raw silks were much higher than those of commercial silkworm strains. Especially, almost equal strength to that of MC502 was observed when MC502 was crossbred with J137, a thin silk-thread producing Chinese silkworm strain C515, and a high-strength silk-thread producing Japanese silkworm strain MN502.
This report describes a method to ascertain the copy-number of transgenes of transgenic silkworm. For this study, probe-based quantitative polymerase chain reaction (probe qPCR) assay using comparative Ct method (ΔΔCt method) was used to detect the piggyBac sequence introduced into the silkworm genome. Using hsp90 gene as a reference, 0–8 copies of the piggyBac gene inserted into the silkworm genome were detected quantitatively.
Silk fibroin (SF) composed of heavy chains (Fib-H), light chains (Fib-L), and fibrohexamerin, is a desirable material for many tissue engineering applications due to its superior moldability and biocompatibility. In this work, we produced transgenic silkworm lines, which express a recombinant protein comprising the basic fibroblast growth factor (bFGF)-binding P7 peptide with homology to bFGF receptors introduced at the C-terminus of the fibroin L-chain, using the piggyBac vector. As a result, one transgenic line 2-23 expressed recombinant fibroin L-chain protein at a relative amount of 26.75 % of all L-chains (which also comprise endogenous L-chains), which indicates high bFGF binding affinity of recombinant SF compared to that of the control (non-transgenic silkworm SF). Our result will contribute to the development of medical application of recombinant SF.
In this study, to develop silk fibroin (SF)-based sponges used in the field of skin esthetics and skin tissue engineering, the influence of the SF solution and the SF sponge on gene expressions in three-dimensional (3-D) reconstructed human keratinocytes with or without melanocytes as a skin equivalent model was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) experiments. Commercially available collagen (Col) solution and sponge were used for comparison. In the 3-D skin model made from keratinocytes, no marked changes in the expressions of genes related to differentiations and barrier functions of epidermis were observed between both solutions and sponges of SF and Col. RT-PCR experiments using the 3-D skin model with both keratinocytes and melanocytes revealed that gene expression levels of melanogenesis-related factors on the 3-D skin models stimulated with the SF sponge were slightly higher than that on the Col sponge. Measurements of pigment areas in the 3-D skin models with melanocytes indicated that almost identical areas of black melanin were detected in the 3-D skin models stimulated with medium, SF sponge, and Col sponge. This study indicates that SF-based materials are biologically inert to human skin epidermis, similar to collagen-based materials.