Abstract
β-lactoglobulin (BLG), a dominant allergen in goat milk, is difficult to remove by traditional biochemical methods. Its elimination from goat milk by genetic modification therefore poses a major challenge for modern goat breeders. A shRNA targeting BLG mRNA with high interference efficiency was identified, with which lentiviral vectors were used for mediating stable shRNA interference in goat-fetal fibroblast cells. Apart from high efficiency in the knockdown of BLG expression in these cells, lentivector-mediated RNAi manifested stable integration into the goat genome itself. Consequently, an in vitro model for goat BLG-content control was compiled, and a goat-cell line for accompanying transgenetic goat production created.
Highlights
Nutritious for humans, especially infants, goat milk and its by-products are considered important allergens in food allergy, whence the restrictions against their wide use in the food industry. b-lactoglobulin (BLG), a major protein in milk (56%-60% of total cow-whey proteins), is considered a dominant allergen (Sharma et al, 2001; Fritsche, 2003).Several biochemical approaches have been applied, in the attempt to reduce the allergenic potential of milk proteins, especially BLG
Individual short hairpin RNA (shRNA) efficiency in inducing RNA interference (RNAi) in Goat embryo fibroblast (GEF) cells was tested in transiently transfected cells
Synchronous with OE-BLG plasmid co-transfection, all the anti-BLG shRNA constructs resulted in a significant reduction (p < 0.05) in the level of BLG mRNA, compared to cells that were only transfected with OE-BLG plasmids, considered as positive control (Figure 2)
Summary
Nutritious for humans, especially infants, goat milk and its by-products are considered important allergens in food allergy, whence the restrictions against their wide use in the food industry. b-lactoglobulin (BLG), a major protein in milk (56%-60% of total cow-whey proteins), is considered a dominant allergen (Sharma et al, 2001; Fritsche, 2003).Several biochemical approaches have been applied, in the attempt to reduce the allergenic potential of milk proteins, especially BLG. Enzymatic hydrolysis, fermentation and glycation (Fritsche, 2003; Hattori et al, 2004; Ehn et al, 2005), as well as the Maillard reaction (Guanhao Bu et al, 2010), are examples Notwithstanding, these always entail increased costs and the accumulation of unexpected by-products. Lentiviral vectors are potent and efficient in enhancing the capacity for stable RNAi mediating-gene silencing in animal cells (Stewart et al, 2003; Xie et al, 2008). This capacity is critical for further application, especially gene knockdown in transgenic animals
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