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Asian-Australas J Anim Sci > Volume 30(8); 2017 > Article
Asian-Australasian Journal of Animal Sciences 2017;30(8): 1175-1182. doi: https://doi.org/10.5713/ajas.16.0697
Human lactoferrin efficiently targeted into caprine beta-lactoglobulin locus with transcription activator-like effector nucleases
Yu-Guo Yuan1,2, Shao-Zheng Song1, Meng-Ming Zhu1, Zheng-Yi He1, Rui Lu1, Ting Zhang1, Fei Mi1, Jin-Yu Wang1,*, Yong Cheng1,2,*
1Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis/College of animal science and technology, Yangzhou University, Yangzhou, Jiangsu 225009, China
2Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Yangzhou, 225001, China
* Corresponding Author: Jin-Yu Wang ,Email: jywang@yzu.edu.cn / Yong Cheng ,Tel: +86-514-87979348, Fax: +86-514-87972218, Email: cheng1391@hotmail.com
Received: September 19, 2016;  Revised: November 11, 2016.  Accepted: December 13, 2016.  Published online December 17, 2016.
Yu-Guo Yuan and Shao-Zheng Song contributed equally to this work.

ABSTRACT
Objective:
To create genetically modified goat as a biopharming source of recombinant human lacotoferrin (hLF) with transcription activator-like effector nucleases.
Method:
TALENs and targeting vector were transferred into cultured fibroblasts to insert hLF cDNA in the goat beta-lactoglobulin (BLG) locus with homology-directed repair. The gene targeted efficiency was checked using sequencing and TE7I assay. The bi-allelic gene targeted colonies were isolated and confirmed with polymerase chain reaction, and used as donor cells for somatic cell nuclear transfer (SCNT).
Results:
The targeted efficiency for BLG gene was approximately 10%. Among 12 Bi-allelic gene targeted colonies, five were used in first round SCNT and 4 recipients (23%) were confirmed pregnant at 30 d. In second round SCNT, 7 (53%), 4 (31%), and 3 (23%) recipients were confirmed to be pregnant by ultrasound on 30 d, 60 d, and 90 d.
Conclusion:
This finding signifies the combined use of TALENs and SCNT can generate bi- allelic knock-in fibroblasts that can be cloned in a fetus. Therefore, it might lay the foundation for transgenic hLF goat generation and possible use of their mammary gland as a bioreactor for large-scale production of recombinant hLF.
Keywords: Beta-lactoglobulin; Gene Targeted; Transcription Activator-Like Effector Nucleases; Human Lacotoferrin; Goat; Nuclear Transfer
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