The flowchart of producing infective BmNPV expressing ten heterologous genes in silkworm larvae or pupae.

<p>The target genes are cloned into the three donor vector (pCTdual, pRADM and pUCDMIG) using usual method. The first two genes carried by pCTdual are inserted into BmBacmid through <i>I-Sce</i> I linearization and <i>red-gam</i> homologous recombination. Four genes in pRADM and the other four genes in pUCDMIG are then introduced into BmBacmid via Tn7 transposition and cre-loxp recombination, respectively. As a result, ten foreign expression cassettes and three antibiotic screening markers, as well as a GFP illumination marker are introduced into BmBacmid. The invasive and DAP auxotrophic <i>E. coli</i> carrying recombinant BmBacmid are injected into silkworm larvae at an appropriate dose. Consequently, recombinant BmNPV will be produced and multiple foreign genes will be expressed in green <i>B. mori</i> larvae or pupae.