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A Pilot Study of Large-Scale Production of Mutant Pigs by ENU Mutagenesis

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A Pilot Study of Large-Scale Production of Mutant Pigs by ENU Mutagenesis 1 A pilot study of large-scale production of mutant pigs by ENU mutagenesis 2 Tang Hai1,8,†, Chunwei Cao1,8,†, Haitao Shang2,8,†, Weiwei Guo4,†,Yanshuang Mu3,8,†, Shulin 3 Yang5,8, Ying Zhang1,8, Qiantao Zheng1,8, Tao Zhang1,8, Xianlong Wang1,8, Yu Liu2,8, Qingran 4 Kong3,8, Kui Li5,8, Dayu Wang1,8, Meng Qi1,8, Qianlong Hong1,8, Rui Zhang1,8, Xiupeng 5 Wang1,8, Qitao Jia1,8, Xiao Wang1,8, Guosong Qin1,8, Yongshun Li1,8, Ailing Luo1,8, Weiwu 6 Jin1,8, Jing Yao1,8, Jiaojiao Huang1,8, Hongyong Zhang1,8, Menghua Li1,8, Xiangmo Xie1,8, 7 Xuejuan Zheng1,8, Kenan Guo2,8, Qinghua Wang2,8, Shibin Zhang2,8, Liang Li2,8, Fei Xie2,8, Yu 8 Zhang3,8, Xiaogang Weng3,8, Zhi Yin3,8, Kui Hu3,8, Yimei Cong3,8, Peng Zheng3,8, Hailong 9 Zou3,8, Leilei Xin5,8, Jihan Xia5,8, Jinxue Ruan5,8, Hegang Li5,8, Weiming Zhao5,8, Jing Yuan5,8, 10 Zizhan Liu5,8, Weiwang Gu6,8, Ming Li6,8, Yong Wang2,8, Hongmei Wang1,9,*, Shiming 11 Yang4,9*, Zhonghua Liu3,9,*, Hong Wei2,9,*, Jianguo Zhao1,9,*, Qi Zhou1,9,*, Anming Meng7,9,* 12 Affiliations: 13 1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese 14 Academy of Sciences, Beijing100101, China, 15 2Department of Laboratory Animal Science, College of Basic Medicine, Third Military 16 Medical University, Chongqing 400038, China, 17 3College of Life Science, Northeast Agricultural University of China, Harbin 150030, China, 18 4Department of Otolaryngology-Head and Neck Surgery, Institute of Otolaryngology, Chinese 19 PLA General Hospital, Beijing 100853, China, 20 5Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, 21 China, 22 6Pearl Laboratory Animal Sci. & Tech. Co. Ltd, Guangzhou 523808, China, 23 7School of Life Sciences, Tsinghua University, Beijing 100084, China, 24 8Chinese Swine Mutagenesis Consortium Working Group, Chinese Swine Mutagenesis 25 Consortium, Beijing, China 26 9Chinese Swine Mutagenesis Consortium Guide Group, Chinese Swine Mutagenesis 27 Consortium, Beijing, China 1 28 29 †These authors contributed equally to this work 30 *Correspondence and requests for materials should be addressed to A. Meng, Q. Zhou, J. Zhao, 31 H. Wei, Z. Liu, H. Wang and S. Yang. 32 [email protected] 33 [email protected] 34 [email protected] 35 [email protected] 36 [email protected] 37 [email protected] 38 [email protected] 39 Competing interests 40 The authors declare that no competing interests exist. 41 42 43 44 45 46 Abstract 47 N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to efficiently generate large scale of 48 mutants and discover genes with novel functions at the whole-genome level in C. elegans, flies, 49 zebrafish and mice, but has never been tried in large model animals. In the current study, we 50 reported a successful systematic three-generation ENU mutagenesis screening in pigs with the 51 establishment of Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs 52 were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were 53 established. The causative mutations in 10 mutant families were further mapped. As examples, 2 54 the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human 55 Mondini dysplasia, and up-regulated expression of FBXO32 is associated with congenital splay 56 legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens 57 an avenue for generating a reservoir of mutants for agriculture production and biomedicine 58 research. 59 60 61 62 63 64 65 Introduction 66 Pigs are an important source of meat worldwide and are widely used in biomedical research 67 (Groenen et al., 2012; Prather, 2013; Wernersson et al., 2005). The similarities of organ size, 68 anatomic and physiologic characteristics and the genome sequence between pigs and humans 69 stimulated the use of these animals as preferred models of various human diseases and sources 70 of allogenetic organs for xenotransplantation. Genetic modifications in pigs are crucial for 71 the generation of tailored disease models, and the knowledge-based development of 72 sustainable pig production relies on the annotation of porcine genomes and the precise and 73 efficient generation of genetic engineering. Although a draft of pig genome was reported in 74 2012 and much progress has been made in pig gene identification, mapping and functional 75 analyses, the current knowledge of the pig functional genome remains limited (Brown and 3 76 Moore, 2012). Understanding the pig genome and identifying the causative genes for certain 77 physiologies are imperative and will not only benefit disease modeling and 78 xenotransplantation but also be invaluable for solving a range of problems associated with 79 pork production (McGonigle and Ruggeri, 2014). 80 ENU-induced mutagenesis is an effective forward genetic approach for identifying 81 functional genes and generating animal models. ENU is a potent mutagen that primarily 82 induces point mutations and chromosome rearrangements in the genome in a random manner 83 (Russel et al., 1979). The advantages of this approach are that no assumptions are needed 84 regarding the underlying genetic causes of physiologic or biologic processes and it allows the 85 precise selection of phenotypes of interest (Acevedo-Arozena et al., 2008). ENU mutagenesis 86 has advantages over knockout strategies in that ENU generates a series of point mutations that 87 frequently mimic the subtlety and heterogeneity of human genetic lesions (Oliver and Davies, 88 2012). In the past, ENU mutagenesis had been used to generate thousands of mutants in C. 89 elegans (De Stasio and Dorman, 2001), flies (Choi et al., 2009; Yu et al., 1987), zebrafish 90 (Driever et al., 1996; Geisler et al., 2007; Haffter et al., 1996; Wienholds et al., 2003; Xiao et 91 al., 2005) and mice (Hrabe de Angelis et al., 2000; Nolan et al., 2000; Vitaterna et al., 1994), 92 leading to the discovery of many important genes and genetic pathways and contributing much 93 to the current understanding of embryonic development, organogenesis and the etiology of 94 various diseases. However, the effectiveness of ENU mutagenesis in large mammalian 95 species remains unknown. The need to annotate functional genomics and generate ideal large 96 animal models prompt us to conduct a forward genetic screen using ENU in pigs. 97 Here, we report the first evidence of the feasibility of the ENU mutagenesis screen in 98 large animals to generate inheritable mutants primarily focused on dysmorphology, growth 99 rate, body weight and blood biochemical parameters. A three-generation breeding scheme 100 was employed for mutation screening, and 36 dominant and 91 recessive novel pig lines were 101 identified. We show that the ENU-induced mutations in various lines can be mapped. These 4 102 findings revealed that ENU mutagenesis in pigs is an efficient strategy to identify genes with 103 novel functions and generate mutants for agricultural production and biomedical research. 104 Results 105 ENU mutagenesis in Bama miniature pigs 106 The Bama miniature pig, a southern Chinese native breed with a body weight of 20-30 kg at 6 107 months of age, was used for mutagenesis in the present study. To determine an appropriate 108 mutagenic but non-toxic dose of ENU (Justice et al., 2000), an ENU concentration of 65 or 85 109 mg/kg bodyweight was intravenously injected three times at weekly intervals into 7- to 110 8-month-old boars (G0) (Figure 1A) (Hrabe de Angelis et al., 2000). The treated boars showed 111 poor-quality sperm at 2-3 weeks after the last injection and underwent a period of azoospermia 112 for 6-8 weeks. G0 boars recovered fertility at 12-14 weeks post-treatment, and there were no 113 obvious differences in sperm quality between the two treatment groups (Figure 114 1-supplement). 115 To evaluate the frequency and mutational pattern of ENU-induced nucleotide changes in 116 pigs, we implemented a trio-based method to assess the ENU-induced mutation frequencies at 117 a genome-wide scale. Two G0 boars, one injected with 65 and the other with 85 mg/kg ENU, 118 were selected, and each G0 produced 5 G1 progenies, each of which derived from a specific 119 ENU mutagenized sperm of G0. Using trio-based 2b-RAD sequencing, the G1 boar specific 120 mutations, absent from the G0 boars and G0 sows, were identified as the ENU-induced 121 mutations. The results indicated that ENU treatment produced a significant number of G to A 122 transitions, and these transitions occurred far more frequently than did transversions (71.2% for 123 transitions versus 28.8% for transversions) (Figure 1B, C and D). An increase of mutation 124 frequency in the 85-mg/kg treatment group was observed compared with the 65-mg/kg 125 treatment group (5.86×10-6 vs. 1.63×10-6) (Supplementary file 1 and Supplementary file 2). 126 The mutation frequency indicated that each G1 pig carried 4500-16,458 heterozygous 127 mutations throughout the genome (pig genome size 2.81×109 bp), most of which presumably 128 were located in the intergenic area (Figure 1E). An archive of DNA, tissues, cells and sperm 5 129 from the 5000 G1 offspring was established to harbor mutations. We concluded that a dose of 130 3×85 mg/kg ENU for three time treatments is suitable for effective mutagenesis in Bama 131 miniature boars. 132 Systematic phenotypic screening for dominant and recessive mutants 133 A three-generation breeding scheme was designed to screen dominant and recessive mutations 134 (Figure 2A). G0 boars were mated with wild-type sows to produce G1 founders, which were 135 observed for novel phenotypes of dominant mutations.
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