Germline Transformation of the Silkworm Bombyx mori L. by Sperm-Mediated Gene Transfer1

Abstract

The domesticated silkworm Bombyx mori L. has important roles in basic biological research and applied science. To explore the practical use of transgenic technology in agricultural silkworm varieties, we fused the neomycin-resistance gene (Neo(R)) and the green fluorescent protein gene (gfp) into the piggyBac-based transposon vector and transduced it into silkworms by sperm-mediated gene transfer (SMGT). Fluorescence observation indicated the positive rate of G0 egg-batches is 72.7%. After screening against the antibiotic G418, development of individual larvae in the same brood showed significant size differences. PCR detection indicated the existence of gfp and Neo(R) and confirmed the positive rate of transgenesis as 0.47%. Southern blot analysis confirmed the presence of the exogenous genes in the genome of G7 larvae. These results show that our strategy is practical and markedly improves the efficiency of SMGT.

Comparison of two methods of sperm‐ and testis‐mediated gene transfer in production of transgenic animals: A systematic review

“…The variety of SMGT hosts reveals the flexibility of this method in sperm‐mediated delivery of foreign DNA into zygotes of a wide range of species, using in vitro fertilization or ICSI. For small animals such as insects, SMGT may be the only viable method, while in the case of externally fertilized animals such as fish, in which sperm are more available and resistant to environmental conditions, SMGT is more accessible and completely reasonable compared to TMGT (Cebeci et al., 2020; Zhou et al., 2012). Mice and rats are the most commonly used species in both SMGT and TMGT studies.…”

“…The other benefits of liposomes include the formation of complexes with both negatively and positively charged molecules, carrying large pieces of DNA, and being targeted to specific cells or tissues (Daraee et al, 2016). Liposomes, either commercial or modified, have been used by approximately 15% of research groups for lipofectione of male germ cells in various organisms, including farm insects, birds, livestock, and laboratory mice (Amaral et al, 2011;Arias et al, 2017;Kim et al, 2019;Samoylov et al, 2013;Zhou et al, 2012). Churchil et al (2011) explored the use of a lipid bilayer nanocapsule as a carrier for gene delivery.…”

Bmovo-1 Regulates Ovary Size in the Silkworm, Bombyx mori

“…Sperm-mediated gene transfer was performed as described by Zhou [21]. The transgenic vector piggyBac-A3GFP-IeNeo-VlgBmovo-1 at 1 µg/µL was mixed at a ratio of 1∶1 with a helper pigA3 vector containing the piggyBac transposase sequence under the control of the A3 promoter.…”