title: Construction and long term preservation of clonal transgenic silkworms using a parthenogenetic strain.
abstract: For the functional analysis of insect genes as well as for the production of recombinant proteins for biomedical use, clonal transgenic silkworms are very useful. We examined if they could be produced in the parthenogenetic strain that had been maintained for more than 40years as a female line in which embryogenesis is induced with nearly 100% efficiency by a heat shock treatment of unfertilized eggs. All individuals have identical female genotype. Silkworm transgenesis requires injection of the DNA constructs into the non-diapausing eggs at the preblastodermal stage of embryogenesis. Since our parthenogenetic silkworms produce diapausing eggs, diapause programing was eliminated by incubating ovaries of the parthenogenetic strain in standard male larvae. Chorionated eggs were dissected from the implants, activated by the heat shock treatment and injected with the transgene construct. Several transgenic individuals occurred in the daughter generation. Southern blotting analysis of two randomly chosen transgenic lines VTG1 and VTG14 revealed multiple transgene insertions. Insertions found in the parental females were transferred to the next generation without any changes in their sites and copy numbers, suggesting that transgenic silkworms can be maintained as clonal strains with homozygous transgenes. Cryopreservation was developed for the storage of precious genotypes. As shown for the VTG1 and VTG14 lines, larval ovaries can be stored in DMSO at the temperature of liquid nitrogen, transferred to Grace's medium during defrosting, and then implanted into larvae of either sex of the standard silkworm strains C146 and w1-pnd. Chorionated eggs, which developed in the implants, were dissected and activated by the heat shock to obtain females (nearly 100% efficiency) or by a cold shock to induce development to both sexes in 4% of the eggs. It was then possible to establish bisexual lines homozygous for the transgene.
authors.affiliation: Biology Centre CAS, 370 05 České Budějovice, Czech Republic. Electronic address: vzabelina@entu.cas.cz.
authors.forename: Valeriya
authors.initials: V
authors.lastname: Zabelina
authors.affiliation: National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan. Electronic address: uchinoK@affrc.go.jp.
authors.forename: Keiro
authors.initials: K
authors.lastname: Uchino
authors.affiliation: Institute of Sericulture, Iikura 1053, 300-0324 Ami-machi, Ibaraki, Japan. Electronic address: mochida@ss.silk.or.jp.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Mochida
authors.affiliation: National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan. Electronic address: yonemura@affrc.go.jp.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Yonemura
authors.affiliation: V.N. Karazin Kharkov National University, 61077 Kharkov, Ukraine. Electronic address: stemway@gmail.com.
authors.forename: Vyacheslav
authors.initials: V
authors.lastname: Klymenko
authors.affiliation: National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Sezutsu
authors.affiliation: National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8634, Japan. Electronic address: ttamura@affrc.go.jp.
authors.forename: Toshiki
authors.initials: T
authors.lastname: Tamura
authors.affiliation: Biology Centre CAS, 370 05 České Budějovice, Czech Republic. Electronic address: sehnal@bc.cas.cz.
authors.forename: František
authors.initials: F
authors.lastname: Sehnal
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D012831:Bombyx / Q000235:genetics*; D015925:Cryopreservation / Q000379:methods*; D047108:Embryonic Development; D005260:Female; D018014:Gene Transfer Techniques; D017344:Genes, Insect; D018869:Heat-Shock Response; D008297:Male; D010312:Parthenogenesis
keywords: Biotechnology; Cryopreservation; Parthenogenesis; Silkworm; Transgenesis