Transgenesis approaches for functional analysis of peptidergic cells in the silkworm Bombyx mori

Abstract

The domestic silkworm, Bombyx mori represents an insect model of great scientific and economic importance. Besides the establishment of a stable germline transformation using the PiggyBac vector, technically feasible methods for in vivo gene delivery and transient gene expression were developed using viral based vectors, especially Sindbis viruses and baculoviruses. The recombinant baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), commonly used for large-scale protein production in permissive cell lines or insects, has been used for foreign gene transfer into specific peptidergic cells of B. mori in vivo. Since targeted gene expression is essential for functional analysis of neuropeptide genes and their receptors, the baculovirus-mediated gene transfer can serve as a reliable approach in reverse genetic studies in the silkworm. We review various strategies employing the baculovirus vector system for transient expression of molecular markers and transcription factors in specific peptidergic cells to investigate their roles in B. mori. We also use this system for functional analysis of neuropeptide signaling in the ecdysis behavioral sequence. Our data indicate that the AcMNPV vector is suitable for efficient delivery of foreign genes and their expression directed into specific peptidergic neurons and endocrine cells of B. mori larvae and pupae. However, some modifications of the vector and steps for optimization are necessary to minimize negative effects of viral infection on the host development. The transient gene expression using the AcMNPV and other virus vectors are promising tools for analysis of molecular mechanisms underlying various neuroendocrine processes during development of B. mori.

Fig. 1

Bi-phasic replication cycle of the AcMNPV. Occlusion derived virions (ODV) released from polyhedra in the alkaline environment of the insect midgut fuse with midgut epithelial cells to trigger primary infection. The viral transcription and replication is initiated upon entering the nucleus. Budding nucleocapsids obtain their final envelope from the host cell membrane enriched in viral glycoproteins. Budded viruses (BV) subsequently start new cycle of infection by entering next susceptible cells through an endocytotic process and spread infection throughout the larva. During the later stages of infection, progeny virus particles become embedded within the matrix of occlusion body protein (polyhedrin). Large numbers of polyhedra or occlusion bodies (OB) accumulate in the host during infection and enter environment upon cell lysis and larval body disintegration to further spread virus infection to surrounding hosts.

Fig. 2

Generation of the recombinant baculovirus using the Bac-to-Bac^® expression system. The recombinant donor vector with a gene of interest under control of the polh promoter is transformed into DH10Bac™competent E. coli cells containing a parent bacmid as well as helper plasmid providing transposition enzymes. Integration of the expression cassette in the baculovirus DNA is achieved by site-specific transposition between the Tn7 elements (Tn7L and Tn7R) of the pFastBac™ donor vector and the mini-attTn7 target site in the bacmid. Since the mini-attTn7 attachment site is inserted in the reading frame of lacZα gene, successful transposition disrupts the expression of LacZα peptide allowing comfortable detection of bacterial colonies containing recombinant bacmid. The extracted recombinant baculovirus DNA is then transfected into cultured insect cells to produce the recombinant baculovirus. Generated recombinant virus particles are subsequently amplified by repeated rounds of infection and after determination of virus titer by plaque assay, the recombinant virus can be used for different purposes. The drawing is modified from the Guide to baculovirus expression vector systems (BEVS) and insect cell culture techniques (Invitrogen Instruction Manual).

Fig. 3

Schematic drawing of the pFastBac plasmid containing a neuropeptide promoter and egfp. The upstream region of a neuropeptide gene is fused with egfp to drive its expression. The promoter-EGFP expression cassette is inserted into a polylinker of the pFastBac-HTB vector (Invitrogen) and used for preparation of the recombinant bacmid DNA.

Fig. 4

Targeted EGFP expression induced by PTTH and ETH promoters using the baculovirus expression system. (A) EGFP expression in two pairs of neurosecretory cells in the brain producing PTTH. (B) EGFP expression in the endocrine Inka cell producing ETH. Scale bars; 50 µm in (A) and 200 µm in (B).