Clonal transgenic silkworms are useful for the functional analysis of insect genes and for the production of recombinant proteins. Such silkworms have previously been created using an existing ameiotic parthenogenetic strain. However, the process was labor intensive, and the efficiency of producing transgenic silkworms was very low. To overcome this issue, we developed a more convenient and efficient method by breeding non-diapausing parthenogenetic strains. The strains produced non-diapausing eggs only when the embryogenesis of the parent eggs was performed at low temperatures, which could then be used for injecting vector plasmids. This demonstrated that transgenic silkworms could be produced with greater ease and efficiency. To breed the strains, we crossed the existing parthenogenetic strains with bivoltine strains and made F1 and F2 from each cross. Then we selected the silkworms whose eggs have a high ability of parthenogenesis and became non-diapausing. We also demonstrated that the germplasm could be cryopreserved in liquid nitrogen. Thus, this method increases the efficiency and ease of using genetically engineered silkworms to analyze gene function and produce recombinant proteins, potentially impacting various industries.

Biology Centre CAS 370 05 České Budějovice Czech Republic

Institute of Agrobiological Sciences NARO Tsukuba Ibaraki 305 8634 Japan

Institute of Sericulture Iikura 1053 300 0324 Ami machi Ibaraki Japan

Ryazan State Radio Engineering University Ryazan 390005 Russia

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The Exact Timing of Microinjection of Parthenogenetic Silkworm Embryos Is Crucial for Their Successful Transgenesis