Targeted Genetic Transformation In Cultured Silkworm Cells Based On PiggyBac Transposon

The genetic transformation technology has been used widely in researches involving gene functions, physiological and biochemical processes, and bioreactor development. However, several problems exist despite of this success, including low integration efficiency of transgenic genes, low expression levels and somehow unpredictable expression patterns of transgenes, and mutation of inherent genes due to the random integration of transgens. One of approaches to resolve these problems is to increase the efficiency of transformation and the level of site-specific integration.The silkworm, Bombyx mori L., is one of the most economic insects and a model for gene function and biological research in insects. So far there have been a large number of reports targeting the genetic transformation of this insect, with very few of them addressing the site-specific transformation. In this study, we conducted some assasys in the silkworm cells, with the purpose of founding some systems that have the potential of realizing site-specifc integrations in this insect.The major results are as follows:1. To find a promoter that can be used to strongly drive expression of piggyBac transposase and thereby increase the transposition efficiceny, eight promoters were compared in cultured silkworm cells (BmN) as to their transcriptional activity. These promoters were hsp70, hsp82, actin3 (A3), polyubiquitin (PUB), a-tubulin, fibroin-L, artificial promoter 3×P3 and immediately early 1 gene promoter flanked by the hr5 enhancer element (hr5-IE1). The results showed that hr5-IE1 displayed the highest transcriptional activity, followed by A3, while the activities of the other six promoters were relatively low. When transfected with an EGFP vector, and a piggyBac helper plasmid in which the expression of piggyBac transposase was driven by hr5-IE1, the EGFP cassette was successfully integrated into the genome of BmN cells.2. By using interplasmid transposition assays, the efficiency of applying a chimeric Gd14-piggyBac transposase to achieve site-specific integration onto a DNA target plasmid was evaluated within silkworm Bm-12 and fruitfly Drosophila S2 cells. The Gal4-piggyBac transposase has a Ga14 DNA binding domain (DBD), and the target plasmid has upstream activating sequences (UAS) to which the Gal4 DBD can bind with high affinity. The results indicated that, in the Bm-12 and S2 cells, the transpositional activity of Ga14-piggyBac transposase was observed to be 3.8- and 4.5-times higher, respectively, compared to the controls absent with Ga14-UAS interaction. Moreover, the Ga14-piggyBac transposase tended to direct the integration of piggyBac element to certain sites of the target plasmid, although the target-directing specificity was not as high as expected.3. The chimeric LexA-piggyBac transposase was evaluated regarding its capacity of increasing transformation efficiceny and site-specific interaction level in the silkworm cell. This fused transposase was supposed to be able to bind a binding-site (BS) sequence which was located on a plasmid DNA molecule to be targeted during interplasmid transpositions. The results showed that, in the Bm-12 cells, the transpositional activity of LexA-piggyBac transposase was 9.0-times higher than the transposase absent with LexA-BS interaction. However, the integrations on the target molecule appeared to be random.4. A silkworm cell line was established which was genetically transformation with EGFP-LexA-BS fragment. This cell line can be used in future studies to further evaluate the capacity of LexA-piggyBac transposase in increasing transformation efficiency and site-specific integration level.The above results suggest that the promoter-enhancer hr5-IE1 has the potential of serving as a sound element in future piggyBac-based transgenic research of Bombyx mori with the capability of increasing transformation efficiency. The chimeric transposases, Ga14-piggyBac and LexA-piggyBac may also serve as approach to increase transformation efficiency. Moreover, the Gd14-piggyBac transposase has the potential of realizing site-directed transgenesis in this insect.