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Report on construction of gene-targeting vector for homologous recombination and transformation in silkworm, Bombyx mori L

Miao, Y.G., Jiang, L.J., Nair, K.S., Chen, Z.W., Ren, S.Z.
Journal of applied entomology 2005 v.129 no.3 pp. 129-133
genetic transformation, green fluorescent protein, fibroins, homologous recombination, gene targeting, Bombyx mori, reporter genes, genetic vectors, transgenic insects
This paper reports the methods of construction of gene-targeting vector for transformation of silkworm, Bombyx mori L. The genomic DNA was isolated from the posterior silk gland of the fifth-instar silkworm larvae. The short fragment (0.5 kb) and long fragment (5 kb) of the fibroin light-chain gene were obtained by polymerase chain reaction (PCR) analysis with special primers and genome DNA as templates and then recombined with pBlueselect vector into pBs-FS-FL. The target green flourescent protein (GFP) gene, was derived from pGEP-1 vector and recombined with pUC19 vector into pUCG vector. GFP was recovered after cutting with restriction endonucleases, PstI and BamHI. Finally, GFP was recombined with pBs-FS-FL into gene-targeting vector, pBs-FS-GFP-FL.