Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer

doi:10.1016/S2095-3119(16)61347-3

Journal of Integrative Agriculture ›› 2016, Vol. 15 ›› Issue (7): 1540-1449.DOI: 10.1016/S2095-3119(16)61347-3

Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer

收稿日期:2015-12-30 出版日期:2016-07-06 发布日期:2016-07-06

Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer

FENG Chong^{1, 2*}, LI Xi-rui^{2, 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

Received:2015-12-30 Online:2016-07-06 Published:2016-07-06
Contact: LUO Yu-zhu, Tel: +86-931-7632483, E-mail: luoyz@gsau.edu.cn; PAN Deng-ke, Tel: +86-10-62815893, Fax: +86-21-64085875, E-mail: pandengke2002@163.com
About author:FENG Chong, E-mail: fengchong81358247@163.com
Supported by:
This work was supported by the National Basic Research Program of China (973 Program) (2015CB554103 and 2011CBA01004).

摘要/Abstract

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.

Key words: transcription activator-like effector nuclease (TALEN) , magnetic beads , somatic cell nuclear transfer (SCNT) , off-target , genetically modified pigs

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

参考文献

Bannister S, Antonova O, Polo A, Lohs C, Hallay N, Valinciute A, Raible F, Tessmar-Raible K. 2014. TALENs mediate efficient and heritable mutation of endogenous genes in the marine annelid Platynereis dumerilii. Genetics, 197, 77–89.

Cade L, Reyon D, Hwang W Y, Tsai S Q, Patel S, Khayter C, Joung J K, Sander J D, Peterson R T, Yeh J R. 2012. Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs. Nucleic Acids Research, 40, 8001–8010.

Camarasa M V, Galvez V M. 2016. Robust method for TALEN-edited correction of pF508del in patient-specific induced pluripotent stem cells. Stem Cell Research & Therapy, 7, 26.

Carlson D F, Tan W, Lillico S G, Stverakova D, Proudfoot C, Christian M, Voytas D F, Long C R, Whitelaw C B, Fahrenkrug S C. 2012. Efficient TALEN-mediated gene knockout in livestock. Proceedings of the National Academy of Sciences of the United States of America, 109, 17382–17387.

Cermak T, Doyle E L, Christian M, Wang L, Zhang Y, Schmidt C, Baller J A, Somia N V, Bogdanove A J, Voytas D F. 2011. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research, 39, e82.

Ding Q, Lee Y K, Schaefer E A, Peters D T, Veres A, Kim K, Kuperwasser N, Motola D L, Meissner T B, Hendriks W T, Trevisan M, Gupta R M, Moisan A, Banks E, Friesen M, Schinzel R T, Xia F, Tang A, Xia Y, Figueroa E, et al. 2013. A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell, 12, 238–251.

Doyle E L, Booher N J, Standage D S, Voytas D F, Brendel V P, Vandyk J K, Bogdanove A J. 2012. TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: Tools for TAL effector design and target prediction. Nucleic Acids Research, 40, W117–122.

Fujimura T, Takahagi Y, Shigehisa T, Nagashima H, Miyagawa S, Shirakura R, Murakami H. 2008. Production of alpha 1,3-galactosyltransferase gene-deficient pigs by somatic cell nuclear transfer: A novel selection method for gal alpha 1,3-Gal antigen-deficient cells. Molecular Reproduction and Development, 75, 1372–1378.

Galili U. 2001. The α-gal epitope (Galα1-3Galβ1-4GlcNAc-R) in xenotransplantation. Biochimie, 83, 557–563.

Hai T, Teng F, Guo R, Li W, Zhou Q. 2014. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Research, 24, 372–375.

Hauschild J, Petersen B, Santiago Y, Queisser A L, Carnwath J W, Lucas-Hahn A, Zhang L, Meng X, Gregory P D, Schwinzer R, Cost G J, Niemann H. 2011. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proceedings of the National Academy of Sciences of the United States of America, 108, 12013–12017.

Huang P, Xiao A, Tong X, Lin S, Zhang B. 2016. Targeted mutagenesis in zebrafish by TALENs. Methods in Molecular Biology, 1338, 191–206.

Jaenisch R, Jahner D, Nobis P, Simon I, Lohler J, Harbers K, Grotkopp D. 1981. Chromosomal position and activation of retroviral genomes inserted into the germ line of mice. Cell, 24, 519–529.

Joziasse D H, Oriol R. 1999. Xenotransplantation: The importance of the Galalpha1,3Gal epitope in hyperacute vascular rejection. Biochimica et Biophysica Acta, 1455, 403–418.

Kang J T, Kwon D K, Park A R, Lee E J, Yun Y J, Ji D Y, Lee K, Park K W. 2015. Production of alpha 1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology. Journal of Veterinary Science, doi: 10.4142/jvs.2016.17.1.89

Li C, Qi R, Singleterry R, Hyle J, Balch A, Li X, Sublett J, Berns H, Valentine M, Valentine V, Sherr C J. 2014. Simultaneous gene editing by injection of mRNAs encoding transcription activator-like effector nucleases into mouse zygotes. Molecular and Cellular Biology, 34, 1649–1658.

Lillico S G, Proudfoot C, Carlson D F, Stverakova D, Neil C, Blain C, King T J, Ritchie W A, Tan W, Mileham A J, McLaren D G, Fahrenkrug S C, Whitelaw C B. 2013. Live pigs produced from genome edited zygotes. Scientific Reports, 3, 2847.

Liu H, Chen Y, Niu Y, Zhang K, Kang Y, Ge W, Liu X, Zhao E, Wang C, Lin S, Jing B, Si C, Lin Q, Chen X, Lin H, Pu X, Wang Y, Qin B, Wang F, et al. 2014. TALEN-mediated gene mutagenesis in rhesus and cynomolgus monkeys. Cell Stem Cell, 14, 323–328.

Moscou M J, Bogdanove A J. 2009. A simple cipher governs DNA recognition by TAL effectors. Science, 326, 1501.

Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T. 2011. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Research, 39, 9283–9293.

Ousterout D G, Perez-Pinera P, Thakore P I, Kabadi A M, Brown M T, Qin X, Fedrigo O, Mouly V, Tremblay J P, Gersbach C A. 2013. Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne muscular dystrophy patients. Molecular Therapy, 21, 1718–1726.

Pan D, Zhang L, Zhou Y, Feng C, Long C, Liu X, Wan R, Zhang J, Lin A, Dong E, Wang S, Xu H, Chen H. 2010. Efficient production of omega-3 fatty acid desaturase (sFat-1)-transgenic pigs by somatic cell nuclear transfer. Science China (Life Sciences), 53, 517–523.

Phelps C J, Koike C, Vaught T D, Boone J, Wells K D, Chen S H, Ball S, Specht S M, Polejaeva I A, Monahan J A, Jobst P M, Sharma S B, Lamborn A E, Garst A S, Moore M, Demetris A J, Rudert W A, Bottino R, Bertera S, Trucco M, et al. 2003. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science, 299, 411–414.

Qiu Z, Liu M, Chen Z, Shao Y, Pan H, Wei G, Yu C, Zhang L, Li X, Wang P, Fan H Y, Du B, Liu B, Liu M, Li D. 2013. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases. Nucleic Acids Research, 41, e120.

Song J, Zhong J, Guo X, Chen Y, Zou Q, Huang J, Li X, Zhang Q, Jiang Z, Tang C, Yang H, Liu T, Li P, Pei D, Lai L. 2013. Generation of RAG 1- and 2-deficient rabbits by embryo microinjection of TALENs. Cell Research, 23, 1059–1062.

Song T, Wu T, Wei F, Li A, Wang F, Xie Y, Liu D, Fan Z, Wang X, Cheng S, Zhang C, He J, Wang S. 2014. Construction of a cDNA library for miniature pig mandibular deciduous molars. BMC Developmental Biology, 14, 16.

Soriano P, Jaenisch R. 1986. Retroviruses as probes for mammalian development: allocation of cells to the somatic and germ cell lineages. Cell, 46, 19–29.

Sung Y H, Baek I J, Kim D H, Jeon J, Lee J, Lee K, Jeong D, Kim J S, Lee H W. 2013. Knockout mice created by TALEN-mediated gene targeting. Nature Biotechnology, 31, 23–24.

Tan W, Carlson D F, Lancto C A, Garbe J R, Webster D A, Hackett P B, Fahrenkrug S C. 2013. Efficient nonmeiotic allele introgression in livestock using custom endonucleases. Proceedings of the National Academy of Sciences of the United States of America, 110, 16526–16531.

Tang C, Zhang Q, Li X, Fan N, Yang Y, Quan L, Lai L. 2014. Targeted modification of CCR5 gene in rabbits by TALEN. Hereditas (Beijing), 36, 360–368. (in Chinese)

Tesson L, Usal C, Menoret S, Leung E, Niles B J, Remy S, Santiago Y, Vincent A I, Meng X, Zhang L, Gregory P D, Anegon I, Cost G J. 2011. Knockout rats generated by embryo microinjection of TALENs. Nature Biotechnology, 29, 695–696.

Veres A, Gosis B S, Ding Q, Collins R, Ragavendran A, Brand H, Erdin S, Cowan C A, Talkowski M E, Musunuru K. 2014. Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell, 15, 27–30.

Wilkie T M, Brinster R L, Palmiter R D. 1986. Germline and somatic mosaicism in transgenic mice. Developmental Biology, 118, 9–18.

Yu S, Luo J, Song Z, Ding F, Dai Y, Li N. 2011. Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle. Cell Research, 21, 1638–1640.

Zhu P, Liu Q, Liu S, Su X, Feng W, Lei X, Liu J, Cui K, Huang B, Shi D. 2015. Generation of foxo3-targeted mice by injection of mRNAs encoding transcription activator-like effector nucleases (TALENs) into zygotes. Reproduction in Domestic Animals, 50, 474–483.

Zu Y, Tong X, Wang Z, Liu D, Pan R, Li Z, Hu Y, Luo Z, Huang P, Wu Q, Zhu Z, Zhang B, Lin S. 2013. TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nature Methods, 10, 329–331.

Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer

Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics