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.

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

“…To date, the piggyBac transposon-based silkworm transgenic technology has been reported to have been successful in functional genomics studies [20][21][22][23][24], silkworm bioreactors [16,17,[25][26][27][28][29], and biomaterials [30,31] and in improving disease resistance [32]. The present results directly confirm these earlier results and establish a practical transgenic technology that can be applied directly to the productive sericulture of silkworm varieties.…”

“…To date, successful SMGT has been reported for a variety of animal classes, including mammals, birds, fish, and insects [11][12][13]. SMGT is a promising technology for the creation of transgenic animals, and reports have appeared of its successful use in silkworm transgenic studies [14][15][16][17]. However, increasing the present low level of efficiency of screening for positive transgenic silkworms is a new challenge.…”

Recent progress in development of transgenic silkworms overexpressing recombinant human proteins with therapeutic potential in silk glands

“…Since then, Tg silkworms have been developed to produce recombinant human proteins, including procollagen (2), cytokines (3,4), and monoclonal antibodies (MoAb) (5-7), in their cocoons and silk glands. The advantages of Tg silkworms producing recombinant human proteins are: i) somewhat larger amounts of recombinant proteins (> 1 mg per larva and/or cocoon) are expressed than those derived from other hosts, ii) physical containment of Tg silkworms is easy because the larvae move slowly and seldom escape and because the moths cannot fly, iii) human pathogens have not been found in Bombyx mori, and iv) year-round breeding is possible with an artificial diet and expanded facilities, reducing the cost of producing recombinant proteins.…”

Delivery of GM-CSF to Protect against Influenza Pneumonia

“…Although the critical disulfide structure is completely conserved between the human and murine growth factors and both have a similar carbohydrate modification pattern, it has been consistently demonstrated that hGM-CSF alone fails to stimulate murine cells and mGM-CSF fails to stimulate human cells [ 21 , 33 – 35 ]. On the other hand there are a few reports on the stimulatory effect of hGM-CSF only on bone marrow cells of irradiated mice in vivo [ 17 , 36 , 37 ] and a report on the healing of the wound surface induced by chemotherapy in mice [ 38 ].…”