Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics and other biomedical areas.

Establishment of efficient genome editing tools is essential for fundamental research, genetic engineering, and gene therapy. Successful construction and application of transcription activator-like effector nucleases (TALENs) in several organisms herald an exciting new era for genome editing. We describe the production of two active TALENs and their successful application in the targeted mutagenesis of silkworm, Bombyx mori, whose genetic manipulation methods are parallel to those of Drosophila and other insects. We will also show that the simultaneous expression of two pairs of TALENs generates heritable large chromosomal deletion. Our results demonstrate that (i) TALENs can be used in silkworm and (ii) heritable large chromosomal deletions can be induced by two pairs of TALENs in whole organisms. The generation and the high frequency of TALENs-induced targeted mutagenesis in silkworm will promote the genetic modification of silkworm and other insect species.