title: Expression of hIGF-I in the silk glands of transgenic silkworms and in transformed silkworm cells.
abstract: To express human insulin-like growth factor-I (hIGF-I) in transformed Bombyx mori cultured cells and silk glands, the transgenic vector pigA3GFP-hIGF-ie-neo was constructed with a neomycin resistance gene driven by the baculovirus ie-1 promoter, and with the hIGF-I gene under the control of the silkworm sericin promoter Ser-1. The stably transformed BmN cells expressing hIGF-I were selected by using the antibiotic G418 at a final concentration of 700-800 microg/mL after the BmN cells were transfected with the piggyBac vector and the helper plasmid. The specific band of hIGF-I was detected in the transformed cells by Western blot. The expression level of hIGF-I, determined by ELISA, was about 7800 pg in 5x10(5) cells. Analysis of the chromosomal insertion sites by inverse PCR showed that exogenous DNA could be inserted into the cell genome randomly or at TTAA target sequence specifically for piggyBac element transposition. The transgenic vector pigA3GFP-hIGF-ie-neo was transferred into the eggs using sperm-mediated gene transfer. Finally, two transgenic silkworms were obtained after screening for the neo and gfp genes and verified by PCR and dot hybridization. The expression level of hIGF-I determined by ELISA was about 2440 pg/g of silk gland of the transgenic silkworms of the G1 generation.
authors.affiliation: Medical College, Soochow University, Suzhou, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhao
authors.forename: Xi
authors.initials: X
authors.lastname: Li
authors.forename: GuangLi
authors.initials: G
authors.lastname: Cao
authors.forename: RenYu
authors.initials: R
authors.lastname: Xue
authors.forename: ChengLiang
authors.initials: C
authors.lastname: Gong
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D012831:Bombyx / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005088:Exocrine Glands / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism*; D008297:Male; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D047011:Silk / Q000378:metabolism*; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D047011:Silk; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I