Abstract
Special value of goat milk in human nutrition and well being is associated with medical problems of food allergies which are caused by milk proteins such as β-lactoglobulin (BLG). Here, we employed transcription activator-like effector nuclease (TALEN)-assisted homologous recombination in goat fibroblasts to introduce human α-lactalbumin (hLA) genes into goat BLG locus. TALEN-mediated targeting enabled isolation of colonies with mono- and bi-allelic transgene integration in up to 10.1% and 1.1%, respectively, after selection. Specifically, BLG mRNA levels were gradually decreasing in both mo- and bi-allelic goat mammary epithelial cells (GMECs) while hLA demonstrated expression in GMECs in vitro. Gene-targeted fibroblast cells were efficiently used in somatic cell nuclear transfer, resulting in production of hLA knock-in goats directing down-regulated BLG expression and abundant hLA secretion in animal milk. Our findings provide valuable background for animal milk optimization and expedited development for agriculture and biomedicine.
Highlights
Human α-lactalbumin, the main whey protein in human milk, accounting for 41% of the whey and 28% of the total protein, has a primary function in regulating the synthesis of lactose which plays an important role in milk production[1, 2]. hLA contains a high proportion of the essential amino acids of tryptophan and cysteine[3, 4]
To extend the use of goat milk as a nutrient for human beings or to “humanize” goat milk, we applied transcription activator-like effector nuclease (TALEN) to knock out by milk proteins such as β-lactoglobulin (BLG) gene and simultaneously increase hLA component to improve the nutritional quality of goat milk, which is unlike the prime pharmaceutical interest to generate animal bioreactors in livestock[17, 18]
Two homology arms flanking hLA-neo cassette were designed for introducing Homology recombination (HR) which can repair the double-strand breaks (DSBs) induced by TALEN pair
Summary
Human α-lactalbumin (hLA), the main whey protein in human milk, accounting for 41% of the whey and 28% of the total protein, has a primary function in regulating the synthesis of lactose which plays an important role in milk production[1, 2]. hLA contains a high proportion of the essential amino acids of tryptophan and cysteine[3, 4]. Goat milk and its by-products of yoghurt, cheese and powder have great significance in human nutrition and are widely appreciated around the world[15, 16]. Several approaches such as heat treatment, enzymatic hydrolysis, fermentation and glycation have been applied to reduce the allergenic potential of BLG protein. These methods are generally cost and may affect other valuable nutrients. To extend the use of goat milk as a nutrient for human beings or to “humanize” goat milk, we applied TALENs to knock out BLG gene and simultaneously increase hLA component to improve the nutritional quality of goat milk, which is unlike the prime pharmaceutical interest to generate animal bioreactors in livestock[17, 18]
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