title: Expression of hGM-CSF in silk glands of transgenic silkworms using gene targeting vector.
abstract: The silk gland of the silkworm is a highly specialized organ that has the wonderful ability to synthesize and secrete silk protein. To express human granucyto-macrophage colony-stimulating factor (hGM-CSF) in the posterior silk glands of gene-targeted silkworms, a targeting vector pSK-FibL-L-A3GFP-PH-GMCSF-LPA-FibL-R was constructed, harboring a 1.2 kb portion of the left homogenous arm (FibL-L), a 0.5 kb portion of the right homogenous arm (FibL-R), fibroin H-chain-promoter-driven hGM-CSF and silkworm actin 3-promoter-driven gfp. The targeting vector was then introduced into the eggs of silkworm, and the transgenic silkworms were verified by PCR and DNA hybridization after being screened for the gfp gene. Western blotting analysis using an antibody against hGM-CSF demonstrated a specific band with a molecular weight of 22 kD in the silk glands of the G3 generation transgenic silkworms. The level of expression of hGM-CSF in the posterior silk glands of the G3 generation transgenic silkworms was approximately 2.70 ng/g of freeze-dried powdered posterior silk gland. These results showed that the heterologous gene could be introduced into the silkworm genome and expressed successfully. Further more, the exogenous genes existing in the G5 transgenic silkworm identified by PCR confirmed its integration stability. In addition, the silk glands containing expressed hGM-CSF performed the function of significantly increasing leukocyte count of CY-treated mice in a time-and-dose-dependent manner.
authors.affiliation: Pre-Clinical Medical and Biological Science College, Soochow University, Suzhou, 215123, China.
authors.forename: Renyu
authors.initials: R
authors.lastname: Xue
authors.forename: Huimei
authors.initials: H
authors.lastname: Chen
authors.forename: Linlin
authors.initials: L
authors.lastname: Cui
authors.forename: Guangli
authors.initials: G
authors.lastname: Cao
authors.forename: Wenlin
authors.initials: W
authors.lastname: Zhou
authors.forename: Xiaojian
authors.initials: X
authors.lastname: Zheng
authors.forename: Chengliang
authors.initials: C
authors.lastname: Gong
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D012831:Bombyx / Q000235:genetics*; D005088:Exocrine Glands / Q000378:metabolism; D005091:Exons; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007958:Leukocyte Count; D051379:Mice; D008813:Mice, Inbred ICR; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor