Skip to main content
SpringerLink
Log in

GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

  • 3 Altmetric

Abstract

Background

Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa.

Methods and results

First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs.

Conclusions

We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Elsea SH, Lucas RE (2002) The mousetrap: what we can learn when the mouse model does not mimic the human disease. ILAR J 43:66–79

    Article  CAS  PubMed  Google Scholar 

  2. Vodicka P, Smetana K Jr, Dvorankova B, Emerick T, Xu YZ, Ourednik J, Ourednik V, Motlik J (2005) The miniature pig as an animal model in biomedical research. Ann N Y Acad Sci 1049:161–171

    Article  PubMed  Google Scholar 

  3. Hay M, Thomas DW, Craighead JL, Economides C, Rosenthal J (2014) Clinical development success rates for investigational drugs. Nat Biotechnol 32:40–51

    Article  CAS  PubMed  Google Scholar 

  4. Bahr A, Wolf E (2012) Domestic animal models for biomedical research. Reprod Domest Anim 47(Suppl 4):59–71

    Article  PubMed  Google Scholar 

  5. Lunney JK, Van Goor A, Walker KE, Hailstock T, Franklin J, Dai C (2021) Importance of the pig as a human biomedical model. Sci Transl Med 13:eabd5758

    Article  CAS  PubMed  Google Scholar 

  6. Hryhorowicz M, Lipinski D, Hryhorowicz S, Nowak-Terpilowska A, Ryczek N, Zeyland J (2020) Application of genetically Engineered Pigs in Biomedical Research.Genes (Basel)11

  7. Imai H, Konno K, Nakamura M, Shimizu T, Kubota C, Seki K, Honda F, Tomizawa S, Tanaka Y, Hata H, Saito N (2006) A new model of focal cerebral ischemia in the miniature pig. J Neurosurg 104:123–132

    PubMed  Google Scholar 

  8. Svendsen O (2006) The minipig in toxicology. Exp Toxicol Pathol 57:335–339

    Article  PubMed  Google Scholar 

  9. Hinrichs A, Renner S, Bidlingmaier M, Kopchick JJ, Wolf E (2021) MECHANISMS IN ENDOCRINOLOGY: transient juvenile hypoglycemia in growth hormone receptor deficiency - mechanistic insights from Laron syndrome and tailored animal models. Eur J Endocrinol 185:R35–R47

    Article  PubMed  Google Scholar 

  10. Iwase H, Ball S, Adams K, Eyestone W, Walters A, Cooper DKC (2021) Growth hormone receptor knockout: relevance to xenotransplantation. Xenotransplantation 28:e12652

    Article  PubMed  Google Scholar 

  11. Zhou Y, Xu BC, Maheshwari HG, He L, Reed M, Lozykowski M, Okada S, Cataldo L, Coschigamo K, Wagner TE, Baumann G, Kopchick JJ (1997) A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc Natl Acad Sci U S A 94:13215–13220

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hinrichs A, Kessler B, Kurome M, Blutke A, Kemter E, Bernau M, Scholz AM, Rathkolb B, Renner S, Bultmann S, Leonhardt H, de Angelis MH, Nagashima H, Hoeflich A, Blum WF, Bidlingmaier M, Wanke R, Dahlhoff M, Wolf E (2018) Growth hormone receptor-deficient pigs resemble the pathophysiology of human Laron syndrome and reveal altered activation of signaling cascades in the liver. Mol Metab 11:113–128

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Cui D, Li F, Li Q, Li J, Zhao Y, Hu X, Zhang R, Li N (2015) Generation of a miniature pig disease model for human Laron syndrome. Sci Rep 5:15603

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Li F, Li Y, Liu H, Zhang X, Liu C, Tian K, Bolund L, Dou H, Yang W, Yang H, Staunstrup NH, Du Y (2015) Transgenic Wuzhishan minipigs designed to express a dominant-negative porcine growth hormone receptor display small stature and a perturbed insulin/IGF-1 pathway. Transgenic Res 24:1029–1042

    Article  CAS  PubMed  Google Scholar 

  15. Kaneko N, Itoh K, Sugiyama A, Izumi Y (2011) Microminipig, a non-rodent experimental animal optimized for life science research: preface. J Pharmacol Sci 115:112–114

    Article  CAS  PubMed  Google Scholar 

  16. Miura N, Kucho K, Noguchi M, Miyoshi N, Uchiumi T, Kawaguchi H, Tanimoto A (2014) Comparison of the genomic sequence of the microminipig, a novel breed of swine, with the genomic database for conventional pig. In Vivo 28:1107–1111

    PubMed  Google Scholar 

  17. Tanihara F, Takemoto T, Kitagawa E, Rao S, Do LTK, Onishi A, Yamashita Y, Kosugi C, Suzuki H, Sembon S (2016) Somatic cell reprogramming-free generation of genetically modified pigs. Sci Adv 2:e1600803

    Article  PubMed  PubMed Central  Google Scholar 

  18. Tanihara F, Hirata M, Nguyen NT, Sawamoto O, Kikuchi T, Doi M, Otoi T (2020) Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes. BMC Biotechnol 20:40

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nguyen TV, Tanihara F, Do LTK, Sato Y, Taniguchi M, Takagi M, Van Nguyen T, Otoi T (2017) Chlorogenic acid supplementation during in vitro maturation improves maturation, fertilization and developmental competence of porcine oocytes. Reprod Domest Anim 52:969–975

    Article  CAS  PubMed  Google Scholar 

  20. Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123

    Article  CAS  PubMed  Google Scholar 

  21. Cradick TJ, Qiu P, Lee CM, Fine EJ, Bao G (2014) COSMID: a web-based Tool for identifying and validating CRISPR/Cas off-target Sites. Mol Ther Nucleic Acids 3:e214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Brinkman EK, Chen T, Amendola M, van Steensel B (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42:e168

    Article  PubMed  PubMed Central  Google Scholar 

  23. Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H, Perry AC (2000) Pig cloning by microinjection of fetal fibroblast nuclei. Science 289:1188–1190

    Article  CAS  PubMed  Google Scholar 

  24. Clement K, Rees H, Canver MC, Gehrke JM, Farouni R, Hsu JY, Cole MA, Liu DR, Joung JK, Bauer DE, Pinello L (2019) CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol 37:224–226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gaj T, Ojala DS, Ekman FK, Byrne LC, Limsirichai P, Schaffer DV (2017) In vivo genome editing improves motor function and extends survival in a mouse model of ALS. Sci Adv 3:eaar3952

    Article  PubMed  PubMed Central  Google Scholar 

  26. Nishio K, Tanihara F, Nguyen TV, Kunihara T, Nii M, Hirata M, Takemoto T, Otoi T (2018) Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos. Reprod Domest Anim 53:313–318

    Article  CAS  PubMed  Google Scholar 

  27. Tanihara F, Hirata M, Nguyen NT, Le QA, Hirano T, Takemoto T, Nakai M, Fuchimoto DI, Otoi T (2018) Generation of a TP53-modified porcine cancer model by CRISPR/Cas9-mediated gene modification in porcine zygotes via electroporation. PLoS ONE 13:e0206360

    Article  PubMed  PubMed Central  Google Scholar 

  28. Tanihara F, Hirata M, Thi Nguyen N, Anh Le Q, Hirano T, Otoi T (2020) Generation of viable PDX1 gene-edited founder pigs as providers of nonmosaics. Mol Reprod Dev 87:471–481

    Article  CAS  PubMed  Google Scholar 

  29. Tanihara F, Hirata M, Nguyen NT, Sawamoto O, Kikuchi T, Otoi T (2021) One-step generation of multiple gene-edited Pigs by Electroporation of the CRISPR/Cas9 system into zygotes to reduce Xenoantigen Biosynthesis.Int J Mol Sci22

  30. Van Vu T, Thi Hai Doan D, Kim J, Sung YW, Thi Tran M, Song YJ, Das S, Kim JY (2021) CRISPR/Cas-based precision genome editing via microhomology-mediated end joining. Plant Biotechnol J 19:230–239

    Article  PubMed  Google Scholar 

  31. McVey M, Lee SE (2008) MMEJ repair of double-strand breaks (director’s cut): deleted sequences and alternative endings. Trends Genet 24:529–538

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Grajcarek J, Monlong J, Nishinaka-Arai Y, Nakamura M, Nagai M, Matsuo S, Lougheed D, Sakurai H, Saito MK, Bourque G, Woltjen K (2019) Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations. Nat Commun 10:4856

    Article  PubMed  PubMed Central  Google Scholar 

  33. Ata H, Ekstrom TL, Martinez-Galvez G, Mann CM, Dvornikov AV, Schaefbauer KJ, Ma AC, Dobbs D, Clark KJ, Ekker SC (2018) Robust activation of microhomology-mediated end joining for precision gene editing applications. PLoS Genet 14:e1007652

    Article  PubMed  PubMed Central  Google Scholar 

  34. Sfeir A, Symington LS (2015) Microhomology-mediated end joining: a back-up survival mechanism or dedicated pathway? Trends Biochem Sci 40:701–714

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Tan J, Zhao Y, Wang B, Hao Y, Wang Y, Li Y, Luo W, Zong W, Li G, Chen S, Ma K, Xie X, Chen L, Liu YG, Zhu Q (2020) Efficient CRISPR/Cas9-based plant genomic fragment deletions by microhomology-mediated end joining. Plant Biotechnol J 18:2161–2163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Coschigano KT, Holland AN, Riders ME, List EO, Flyvbjerg A, Kopchick JJ (2003) Deletion, but not antagonism, of the mouse growth hormone receptor results in severely decreased body weights, insulin, and insulin-like growth factor I levels and increased life span. Endocrinology 144:3799–3810

    Article  CAS  PubMed  Google Scholar 

  37. Liu JL, Coschigano KT, Robertson K, Lipsett M, Guo Y, Kopchick JJ, Kumar U, Liu YL (2004) Disruption of growth hormone receptor gene causes diminished pancreatic islet size and increased insulin sensitivity in mice. Am J Physiol Endocrinol Metab 287:E405–E413

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Dr. Shota Nakade (Massachusetts Institute of Technology) for deep sequencing analysis using CRISPResso. We also thank the Nippon Food Packer, K. K. Shikoku (Tokushima, Japan), for supplying pig ovaries. We acknowledge Tokushima University for providing financial support through the Research Clusters program of Tokushima University. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Faculty of Bioscience and Bioindustry, Tokushima University, Ishii, Myozai-gun, Tokushima, 7793233, Japan

    Fuminori Tanihara, Maki Hirata, Zhao Namula, Lanh Thi Kim Do, Qingyi Lin, Koki Takebayashi, Megumi Nagahara & Takeshige Otoi

  2. Bio-Innovation Research Center, Tokushima University, Ishii, Myozai-gun, Tokushima, 7793233, Japan

    Maki Hirata, Qingyi Lin, Koki Takebayashi, Megumi Nagahara & Takeshige Otoi

  3. College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang, 524088, China

    Zhao Namula

  4. Faculty of Veterinary Science, Prince of Songkla University, Songkhla, 90110, Thailand

    Manita Wittayarat

  5. Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi, 100000, Vietnam

    Lanh Thi Kim Do

  6. Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi, 3290498, Japan

    Hiromasa Hara

  7. Laboratory of Regenerative And Cellular Medicine, Jichi Medical University, Tochigi, 3290498, Japan

    Hiromasa Hara

Authors
  1. Fuminori Tanihara
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maki Hirata
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhao Namula
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Manita Wittayarat
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lanh Thi Kim Do
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qingyi Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Koki Takebayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hiromasa Hara
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Megumi Nagahara
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Takeshige Otoi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Fuminori Tanihara, Maki Hirata, and Takeshige Otoi contributed to the study conception and design. Material preparation, data collection and analysis were performed by Fuminori Tanihara, Maki Hirata, Zhao Namula, Manita Wittayarat, Lanh Thi Kim Do, Qingyi Lin, Koki Takebayashi, Hiromasa Hara, and Megumi Nagahara. The first draft of the manuscript was written by Fuminori Tanihara and Manita Wittayarat revised the manuscript. All authors commented on previous versions of the manuscript. Takeshige Otoi supervised this study, and edited the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Takeshige Otoi.

Ethics declarations

Conflict of interest

The authors declare that there is no conflict of interest.

Ethics approval

The Institutional Animal Care and Use Committee of Tokushima University approved the animal experiments in the present study (approval number: T2019-11).

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tanihara, F., Hirata, M., Namula, Z. et al. GHR-mutant pig derived from domestic pig and microminipig hybrid zygotes using CRISPR/Cas9 system. Mol Biol Rep 50, 5049–5057 (2023). https://doi.org/10.1007/s11033-023-08388-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-023-08388-3

Keywords

Search

Navigation