title: Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.
abstract: Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription-activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors: Yan
authors: Y
authors: Luo
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors: Yongsheng
authors: Y
authors: Wang
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors: Jun
authors: J
authors: Liu
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors: Chenchen
authors: C
authors: Cui
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors: Yongyan
authors: Y
authors: Wu
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors: Hui
authors: H
authors: Lan
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors: Qi
authors: Q
authors: Chen
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors: Xu
authors: X
authors: Liu
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors: Fusheng
authors: F
authors: Quan
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors: Zekun
authors: Z
authors: Guo
authors: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China.|Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
authors: Yong
authors: Y
authors: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005091:Exons; D005260:Female; D056426:Genetic Loci; D006801:Humans; D007782:Lactoglobulins / Q000235:genetics; D008321:Mammary Glands, Animal / Q000378:metabolism*; D008892:Milk / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism*; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics* / Q000378:metabolism
chemical_list: D007782:Lactoglobulins; D011994:Recombinant Proteins; D012709:Serum Albumin; D000069896:Transcription Activator-Like Effector Nucleases