Elementary research into the transformation BmN cells mediated by the piggyBac transposon vector

Abstract

To generate stable transformants of Bombyx mori silkworm BmN cells continuously expressing a target gene from a piggyBac-derived vector, BmN cells were transfected with a piggyBac vector containing a neomycin-resistance gene, green fluorescent protein gene, and human insulin-like growth factor I gene (hIGF-I) and a helper plasmid containing the piggyBac transposase sequence under the control of the B. mori actin 3 (A3) promoter. With the antibiotic G418, we selected stably transformed BmN cells expressing hIGF-I from the piggyBac-derived vector containing a neomycin-resistance gene driven by the ie-1 promoter from the B. mori nucleopolyhedrovirus. However, no stably transformed BmN cells transformed with the piggyBac element vector containing an SV40-promoter-driven neomycin-resistance gene were isolated. Determined with an enzyme-linked immunosorbent assay, the expression level of hIGF1 was about 7.8 ng in 5 × 10 5 cells in which the h IGF-I gene was driven by the sericin-1 promoter, and 147.5 ng in 5 × 10 5 cells in which the h IGF-I was under control of B. mori fibroin heavy chain gene ( fib-H) promoter with its downstream signal peptide sequence. Analysis of the chromosomal insertion site by inverse PCR showed that the exogenous DNA was inserted into the cell genome randomly or at a TTAA target sequence, characteristic of piggyBac element transposition. These results are particularly important because piggyBac has been suggested for use in the transgenesis of silkworm cells.