Journal
INSECT SCIENCE
Volume 28, Issue 5, Pages 1277-1289Publisher
WILEY
DOI: 10.1111/1744-7917.12866
Keywords
inducible gene expression; PhiC31integrase; promoter flipping; silkworm
Categories
Funding
- National Natural Science Foundation of China [31530071]
- Chongqing Science and Technology Commission [CSTC2018JCYJAX0298]
- Fundamental Research Funds for the Central Universities [XDJK2018C064]
- State Key Laboratory of Silkworm Genome Biology [SKLSGB1819-1]
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The phiC31 integrase-mediated FLIP system allows inducible expression of target genes in silkworm. The successful construction of binary transgenic silkworm lines using FLIP and phiC31-NLS integrase systems resulted in high-level expression of DsRed in silkworm offspring. This provides an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.
Inducible gene-expression systems play important roles in gene functional assays in the post-genome era.Streptomycesphage-derived phiC31 integrase, which mediates an irreversible site-specific cassette exchange between the phage attachment site (attP) and the bacterial attachment site (attB), provides a promising option for the construction of a controllable gene-expression system. Here, we report a phiC31 integrase-mediated promoter flip system (FLIP) for the inducible expression of target genes in silkworm (Bombyx mori). First, we constructed a FLIP reporter system, in which a BmAct4 promoter with enhanced translational efficiency was flanked by theattBandattPsites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene. The coexpression of a C-terminal modified phiC31-NLS integrase carrying a simian virus 40 (SV40) nuclear localization signal (NLS) effectively flipped the BmAct4 promoter through anattB/attPexchange, thereby activating the downstream expression ofDsRedin a silkworm embryo-derived cell line, BmE. Subsequently, the FLIP system, together with a system continuously expressing the phiC31-NLS integrase, was used to construct binary transgenic silkworm lines. Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter, with an approximately 39% heritable transformation efficiency in silkworm offspring, leading to the constitutive and high-level expression of DsRed in silkworms, which accounted for approximately 0.81% of the silkworm pupal weight. Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.
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