Generation of ETV5 Knockout Pigs with CRISPR/Cas9

DOI: 10.18805/IJAR.B-1327    | Article Id: B-1327 | Page : 999-1004
Citation :- Generation of ETV5 Knockout Pigs with CRISPR/Cas9.Indian Journal of Animal Research.2021.(55):999-1004
Mao Zhang, Gengyuan Cai, Rong Zhou, Huaqiang Yang 281943141@qq.com
Address : National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou-510 642, P.R. China.
Submitted Date : 27-09-2020
Accepted Date : 4-01-2021


Background: Ets variant factor 5 (ETV5) plays an important regulatory role in mouse Spermatogonial stem cells (SSCs) self-renewal. ETV5 knockout (KO) mice exhibit a progressive loss of SSCs and resulting in a Sertoli cell-only phenotype. The current study was aimed to use gene editing technology to obtain ETV5-KO pigs as a model for studying the apoptosis mechanism of SSCs and further clarify the function of ETV5 gene in pigs.
Methods: A gene editing plasmid for the porcine ETV5 gene was constructed, transfected into porcine fetal fibroblasts by electroporation to obtain ETV5-KO cells. ETV5-KO cells were used as donors to prepare ETV5-KO pigs by somatic cell nuclear transfer (SCNT). Testis tissues were collected for hematoxylin and eosin (HE), immunohistochemistry (IHC), RT-PCR testing and blood for ELISA testing from ETV5-KO pig.
Result: In the present study, we used the CRISPR/Cas9 system and SCNT to generate homozygous ETV5-KO pigs. We observed 3 phenotypes in these pigs: normal testis development after birth, the SSCs in the seminiferous tubules did not show obviously extinction at sexual maturity and normal spermatogenesis.




  1. Chen, C., Ouyang, W., Grigura, V., Zhou, Q., Carnes, K., Lim, H., Zhao, G., Arber, S., Kurpios, N., Murphy, T.L., Cheng, A.M., Hassell, J.A., Chandrashekar, V., Hofmann, M., Hess, R.A. and Murphy, K.M. (2005). ERM is required for transcriptional control of the spermatogonial stem cell niche. Nature. 436(7053): 1030-1034.
  2. Eo, J., Shin, H., Kwon, S., Song, H., Murphy, K.M. and Lim, H.J. (2011). Complex ovarian defects lead to infertility in Etv5-/- female mice. Molecular Human Reproduction. 17(9): 568-576.
  3. Jamsai, D., Clark, B.J., Smith S.J., Whittle, B., Goodnow, C.C. and M.K.O.B. Christopher J. Ormandy. (2013). A Missense Mutation in the Transcription Factor ETV5 Leads to Sterility, Increased Embryonic and Perinatal Death, Postnatal Growth Restriction, Renal Asymmetry. Plos one. 10(8): e77311.
  4. Harshini, V., Devi, K.S., Kumari, B.P. and Suresh, J. (2018). A comparative study on cytogenetic profile of Large White Yorkshire crossbred and non-descript pigs. Indian Journal of Animal Research. 53(1): 8-13.
  5. Luo, J., Megee, S., Rathi, R. and Dobrinski, I. (2006). Protein gene product 9.5 is a spermatogonia-specific marker in the pig testis: Application to enrichment and culture of porcine spermatogonia. Molecular Reproduction and Development. 73(12): 1531-1540.
  6. Mehta, P., Sharma, A. and Kaushik, R. (2017). Transgenesis in farm animals-A review. Agricultural Reviews. 38: 129-36
  7. Park, K., Kaucher, A.V., Powell, A., Waqas, M.S., Sandmaier, S.E.S., Oatley, M.J., Park, C., Tibary, A., Donovan, D.M., Blomberg, L.A., Lillico, S.G., Whitelaw, C.B.A., Mileham, A., Telugu, B.P. and Oatley, J.M. (2017). Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene. Scientific Reports. 7: 40176.
  8. Sato, T., Katagiri, K., Gohbara, A., Inoue, K., Ogonuki, N., Ogura, A., Kubota, Y. and Ogawa, T. (2011). In vitro production of functional sperm in cultured neonatal mouse testes. Nature. 471(7339): 504-507.
  9. Sato, T., Katagiri, K., Yokonishi, T., Kubota, Y., Inoue, K., Ogonuki, N., Matoba, S., Ogura, A. and Ogawa, T. (2011). In vitro production of fertile sperm from murine spermatogonial stem cell lines. Nature Communications. 2: 1-7.
  10. Schlesser, H.N., Simon, L., Hofmann, M., Murphy, K.M., Murphy, T., Hess, R.A. and Cooke, P.S. (2008). Effects of ETV5 (Ets Variant Gene 5) on Testis and Body Growth, Time Course of Spermatogonial Stem Cell Loss and Fertility in Mice1. Biology of Reproduction. 78(3): 483-489.
  11. Sharma, A., Shah, S.M., Saini, N., Kaushik, R., Singh, M.K., Manik, R.S., Singla, S.K., Palta, P. and Chauhan, M.S. (2015). Derivation, enrichment and characterization of goat (Capra hircus) spermatogonial stem cells from pre-pubertal testes. Indian Journal of Animal Research. 50(5): 662-667.
  12. Tyagi, G., Carnes, K., Morrow, C., Kostereva, N.V., Ekman, G.C., Meling, D.D., Hostetler, C., Griswold, M., Murphy, K.M., Hess, R.A., Hofmann, M. and Cooke, P.S. (2009). Loss of Etv5 Decreases Proliferation and RET Levels in Neonatal Mouse Testicular Germ Cells and Causes an Abnormal First Wave of Spermatogenesis1. Biology of Reproduction. 81(2): 258-266.
  13. Yang, R., Dong, L., Liu, S., Cheng, Y., Wei, W., Song, J., Li, H., Geng, H., Hao, L. and Zhang, Y. (2019). Novel alternative splice variant of IGF-1R and its mRNA expression patterns in BaMa and Landrace pigs. Indian J. Anim. Res. 1-6.
  14. Zheng, Q., Lin, J., Hambly, C., Qin, G., Yao, J., Song, R., Jia, Q., Wang, X., Li, Y., Zhang, N., Piao, Z., Ye, R., Speakman, O.R., Wang, H., Zhou, Q., Wang, Y., Jin, W. and Zhao, J. (2017). Reconstitution of UCP1 using CRISPRCas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity. Proceedings of the National Academy of Sciences of the United States of America: E9474-E9482.
  15. Zheng, Y., Zhang, Y., Qu, R., He, Y., Tian, X. and Zeng, W. (2014) Spermatogonial stem cells from domestic animals:progress and prospects. Reproduction. 147(3): R65-R74.
  16. Zhou, X., Xin, J., Fan, N., Zou, Q., Huang, J., Ouyang, Z., Zhao, Y., Zhao, B., Liu, Z., Lai, S., Yi, X., Guo, L., Esteban, M.A., Zeng, Y., Yang, H. and Lai, L. (2015). Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cellular and Molecular Life Sciences. 72(6): 1175-1184.

Global Footprints