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Efficient generation of GHR knockout Bama minipig fibroblast cells using CRISPR/Cas9-mediated gene editing

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Abstract

Dwarfism, also known as growth hormone deficiency (GHD), is a disease caused by genetic mutations that result in either a lack of growth hormone or insufficient secretion of growth hormone, resulting in a person’s inability to grow normally. In the past, many studies focusing on GHD have made use of models of other diseases such as metabolic or infectious diseases. A viable GHD specific model system has not been used previously, thus limiting the interpretation of GHD results. The Bama minipig is unique to Guangxi province and has strong adaptability and disease resistance, and an incredibly short stature, which is especially important for the study of GHD. In addition, studies of GHR knockout Bama minipigs and GHR knockout Bama minipig fibroblast cells generated using CRISPR/Cas9 have not been previously reported. Therefore, the Bama minipig was selected as an animal model and as a tool for the study of GHD in this work. In this study, a Cas9 plasmid with sgRNA targeting the first exon of the GHR gene was transfected into Bama minipig kidney fibroblast cells to generate 22 GHR knockout Bama minipig kidney fibroblast cell lines (12 male monoclonal cells and 10 female monoclonal cells). After culture and identification, 11 of the 12 male clone cell lines showed double allele mutations, and the rate of positive alteration of GHR was 91.67%. Diallelic mutation of the target sequence occurred in 10 female clonal cell lines, with an effective positive mutation rate of 100%. Our experimental results not only showed that CRISPR/Cas9 could efficiently be used for gene editing in Bama minipig cells but also identified a highly efficient target site for the generation of a GHR knockout in other porcine models. Thus, the generation of GHR knockout male and female Bama fibroblast cells could lay a foundation for the birth of a future dwarfism model pig. We anticipate that the “mini” Bama minipig will be of improved use for biomedical and agricultural scientific research and for furthering our understanding of the genetic underpinnings of GHD.

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Acknowledgments

We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

Funding

This work was financially supported by the Guangxi Special Project for Culturing Academic/Technical Leaders of New Century (Year 2016).

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Author notes

  1. Rui Wang and Jian-Ying Zhang contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources; Guangxi High Education Key Laboratory for Animal Reproduction and Biotechnology; Faculty of Animal Science & Technology, Guangxi University, Nanning, 530004, China

    Rui Wang, Jian-Ying Zhang, Ke-Huan Lu, Sheng-Sheng Lu & Xiang-Xing Zhu

  2. Guangdong Center of Gene Editing Engineering, Foshan University, Foshan, 528000, China

    Xiang-Xing Zhu

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  1. Rui Wang
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Corresponding authors

Correspondence to Sheng-Sheng Lu or Xiang-Xing Zhu.

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All animal procedures used in this study complied with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Guangxi University.

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The authors declare that they have no conflict of interest.

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Editor: Tetsuji Okamoto

Electronic supplementary material

Supplementary Fig S1

Generation of GHR knockout female Bama minipig kidney fibroblast cells. Genomic indel details of CRISPR/Cas9-edited female Bama minipig kidney fibroblast cell colonies. Upward triangles represent deletions and WT means wild type control. The sgRNA sequence is marked with lines. Protospacer adjacent motif (PAM) sequences are marked in blue. The predicted cleavage site is indicated by the blue arrowhead. M means DNA marker. (PNG 5799 kb)

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Wang, R., Zhang, JY., Lu, KH. et al. Efficient generation of GHR knockout Bama minipig fibroblast cells using CRISPR/Cas9-mediated gene editing. In Vitro Cell.Dev.Biol.-Animal 55, 784–792 (2019). https://doi.org/10.1007/s11626-019-00397-6

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  • DOI: https://doi.org/10.1007/s11626-019-00397-6

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