Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination.
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.
College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Hongmei
H
Zhu
College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Jun
J
Liu
College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Chenchen
C
Cui
College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Yujie
Y
Song
College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Hengtao
H
Ge
College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Linyong
L
Hu
College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Qian
Q
Li
College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Yaping
Y
Jin
College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.
Yong
Y
Zhang
D000483:Alleles; D000818:Animals; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006041:Goats; D059765:Homologous Recombination; D006801:Humans; D007768:Lactalbumin / Q000235:genetics*; D007782:Lactoglobulins / Q000235:genetics*; D008892:Milk / Q000378:metabolism; D016254:Mutagenesis, Insertional; D053652:Nuclear Transfer Techniques; D010053:Ovary / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D000069896:Transcription Activator-Like Effector Nucleases / Q000737:chemistry*
D007782:Lactoglobulins; D007768:Lactalbumin; D000069896:Transcription Activator-Like Effector Nucleases