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Journal of Integrative Agriculture  2016, Vol. 15 Issue (7): 1540-1449    DOI: 10.1016/S2095-3119(16)61347-3
Animal Science · Veterinary Science Advanced Online Publication | Current Issue | Archive | Adv Search |
Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer
FENG Chong1, 2*, LI Xi-rui2, 4*, CUI Hui-ting3, LONG Chuan2, LIU Xia1, TIAN Xing-hua4, PAN Deng-ke2, LUO Yu-zhu1
1 Gansu Key Laboratory of Herbivores Animal Biotechnology, College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, P.R.China
2 Key Laboratory Farm Animal Genetic Resources and Germplasm Innovation, Ministry of Agriculture/Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
3 State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen 361102, P.R.China
4 Laboratory of Biochemistry and Molecular Biology, College of Life Science, Henan University, Kaifeng 475000, P.R.China
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Abstract      The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cell nuclear transfer (SCNT) method has been successfully applied for creating genetically modified pigs. However, methods for isolating cells with biallelic indels requires further improvement because of the relatively low enrichment efficiency of mutated somatic cells. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modified pigs. In this study, an efficient method to increase the enrichment efficiency of TALEN-mediated biallelic knockout (KO) cells was established, and corresponding genetically modified pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase (GGTA1) gene locus. TALEN mRNA was transfected into the ear ?broblasts followed by the enrichment of α-Gal null cells of minipigs using isolectin B4 (IB4) lectin and magnetic beads. A total of 115 cell colonies were formed and validated to be GGTA1 KO cells by sequencing and 10 biallelic KO cell colonies were used as nuclear donors for SCNT. Thirty GGTA1 biallelic KO piglets were successfully delivered and grew normally. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modified pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing genetically modified pig lines with expected genotypes for agricultural breeding and biomedical application.
Keywords:  transcription activator-like effector nuclease (TALEN)        magnetic beads        somatic cell nuclear transfer (SCNT)        off-target        genetically modified pigs  
Received: 30 December 2015   Accepted:

This work was supported by the National Basic Research Program of China (973 Program) (2015CB554103 and 2011CBA01004).

Corresponding Authors:  LUO Yu-zhu, Tel: +86-931-7632483, E-mail:; PAN Deng-ke, Tel: +86-10-62815893, Fax: +86-21-64085875, E-mail:   
About author:  FENG Chong, E-mail:

Cite this article: 

FENG Chong, LI Xi-rui, CUI Hui-ting, LONG Chuan, LIU Xia, TIAN Xing-hua, PAN Deng-ke, LUO Yuzhu. 2016. Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer. Journal of Integrative Agriculture, 15(7): 1540-1449.

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