ABSTRACT
Diapause is an important biological characteristic for many insect species to adapt to adverse environmental conditions and maintain the continuity of the race. Compared with the traditional hydrochloric acid or/and cold storage treatment methods, the artificial corona incubation technology of silkworm (Bombyx mori) eggs has many advantages including, the absence of pollution, easy operation and safety. However, this technology has not yet been applied in sericulture. In this study, we developed a novel artificial corona instrument to successfully disrupt the diapause of newly laid and refrigerated eggs from various Chinese and Japanese lineage silkworm strains. Subsequently, we invented a very early corona treatment (VECT) strategy to prevent the diapause of newly laid silkworm eggs within 4 h of oviposition. The hatching rates of the larvae were more than 95% in all diapause silkworm strains, which was comparable to the effect of the traditional HCl treatment strategy. In addition, we developed a combination strategy of VECT and pre-blastoderm microinjection and successfully created transgenic silkworms in various diapause strains. The results of the current study can aid in improving the corona artificial incubation technology and promote its application in sericulture.
ABSTRACT
A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.
Subject(s)
Bombyx/genetics , DNA Nucleotidyltransferases/metabolism , Recombination, Genetic , Animals , Animals, Genetically Modified , Base Sequence , Bombyx/embryology , Embryo, Nonmammalian/metabolism , Gene Expression , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Genome, Insect/genetics , Green Fluorescent Proteins/genetics , Injections , Luminescent Proteins/genetics , Molecular Sequence Data , Mutagenesis, Insertional/genetics , Reproducibility of ResultsABSTRACT
Cre/lox system derived from P1 bacteriaphage can quickly and effectively achieve gene insertion, deletion, replacement, and inversion by means of site-specific recombination. As one of the most important tools for gene targeting at present, Cre/lox system has been widely used in Arabidopsis thaliana, Oryza sativa L., Mus musculus, Drosophila melanogaster, Danio rerio, and other higher eukaryotic organisms. This review roundly described the basic profile of Cre/lox system, and its application in higher eukaryotes. In addition, we also discussed the main problems and developmental trend of the Cre/lox system in this review, which can be a good reference for using Cre/lox system to realize the gene manipulations of the different high eukaryotic organisms.
