Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination

Hongmei Zhu; Jun Liu; Chenchen Cui; Yujie Song; Hengtao Ge; Linyong Hu; Qian Li; Yaping Jin; Yong Zhang

doi:10.1371/journal.pone.0156636

Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination

PLoS One. 2016 Jun 3;11(6):e0156636. doi: 10.1371/journal.pone.0156636. eCollection 2016.

Authors

Hongmei Zhu^{1

2}, Jun Liu^{1

2}, Chenchen Cui^{1

2}, Yujie Song^{1

2}, Hengtao Ge^{1

2}, Linyong Hu^{1

2}, Qian Li^{1

2}, Yaping Jin^{1

2}, Yong Zhang^{1

2}

Affiliations

¹ College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.
² Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, 712100, Shaanxi, China.

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.

MeSH terms

Alleles
Animals
Female
Fibroblasts / metabolism
Goats
Homologous Recombination*
Humans
Lactalbumin / genetics*
Lactoglobulins / genetics*
Milk / metabolism
Mutagenesis, Insertional*
Nuclear Transfer Techniques
Ovary / metabolism
Plasmids / metabolism
Transcription Activator-Like Effector Nucleases / chemistry*

Substances

Lactoglobulins
Lactalbumin
Transcription Activator-Like Effector Nucleases

Grants and funding

This work is financially supported by the China National Program for Transgenic Animal (2014ZX08008-004) and the National High Technology Research and Development Program of China (2011AA100303). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.