Generation of global Spata19 knockout mouse using CRISPR/Cas9 nickase technology

authors:

avatar Mahsa Zargar , avatar Abbas Jamshidizad , avatar Aidin RahimTayefeh , avatar Ehsan Hashemi , avatar Ali Najafi , avatar Mehdi Shamsara , * , avatar mohammad hossein modarressi ORCID

Koomesh: Vol.22, issue 3; 380-388
published online: September 02, 2020
article type: Research Article
received: June 02, 2019
accepted: January 02, 2020

how to cite: Zargar M, Jamshidizad A, RahimTayefeh A, Hashemi E, Najafi A, et al. Generation of global Spata19 knockout mouse using CRISPR/Cas9 nickase technology. koomesh. 2020;22(3):e153192. 

Abstract

Introduction: SPATA19 gene is expressed in developmental stages of testis and some organs, but so far its function has only been examined in the testis. In this study, we provided an effective pathway for the generation of these mice using new CRISPR / Cas9 nickase method while generating Spata19 knockout mice for future studies in other organs. Materials and Methods: CRISPR / Cas9 nickase plasmids were synthesized using pX335 vector and designed gRNAs. So, each plasmid was transformed into E.coli and clonal selection was performed. The plasmids extracted from the bacteria were mixed in equal proportions and injected into the male pronucleus. Correspondingly, the zygotes carrying the plasmid were transferred to Oviduct of Foster mice. After pregnancy extent, born mice were analyzed. Results: We generated global knockout mice using CRISPR/Cas9 nickase technology. This deletion was examined at three levels of DNA, RNA and protein. Generated 2 nucleotides deletion was confirmed by sequencing at three levels of DNA, RNA and protein. By translating the mutated RNA sequence, it was determined that this deletion could cause premature stop codon. Phenotypic analysis confirmed infertility of male homozygous mice (Spata19 -/-). Conclusion: By generating Spata19 knockout mice, we present a protocol for generation of knockout mice. These mice could be applied as the infertile male mice and also for further characterization of Spata19 gene.  

References

  • 1.

    Nourashrafeddin S, EbrahimzadehVesal R, Miryounesi M, Aarabi M, Zarghami N, Modarressi MH, Nouri M. Analysis of SPATA 19 gene expression during male germ cells development, lessons from in vivo and in vitro study. Cell Biol Int Rep 2014; 21: 1-7.

  • 2.

    Doiguchi M, Yamashita H, Ichinose J, Mori T, Shibata Y, Iida H. Complementary DNA cloning and characterization of rat spergen-1, a spermatogenic cell-specific gene-1, containing a mitochondria-targeting signal. Biol Reprod 2002; 66: 1462-1470.

  • 3.

    Babatunde KA, Najafi A, Salehipour P, Modarressi MH, Mobasheri MB. Cancer/Testis genes in relation to sperm biology and function. Iran J Basic Med Sci 2017; 20: 967-974.

  • 4.

    Suzuki-Toyota F, Ito C, Toyama Y, Maekawa M, Yao R, Noda T, et al. Factors maintaining normal sperm tail structure during epididymal maturation studied in Gopc/ mice. Biol Reprod 2007; 77: 71-82.

  • 5.

    Mi Y, Shi Z, Li J. Spata19 is critical for sperm mitochondrial function and male fertility. Mol Reprod Dev 2015; 82: 907-913.

  • 6.

    Ghafouri-Fard S, Ousati Ashtiani Z, Sabah Golian B, Hasheminasab SM, Modarressi MH. Expression of two testis-specific genes, SPATA19 and LEMD1, in prostate cancer. Arch Med Res 2010; 41: 195-200.

  • 7.

    Ghafouri-Fard S, Abbasi A, Moslehi H, Faramarzi N, Taba Taba Vakili S, Mobasheri MB, Modarressi MH. Elevated expression levels of testisspecific genes TEX101 and SPATA19 in basal cell carcinoma and their correlation with clinical and pathological features. Br J Dermatol 2010; 162: 772-779.

  • 8.

    Jansen IE, Gibbs JR, Nalls MA, Price TR, Lubbe S, van Rooij J, et al. Establishing the role of rare coding variants in known Parkinson's disease risk loci. Neurobiol Aging 2017; 59: 220. e11-220.

  • 9.

    Szabo L, Morey R, Palpant NJ, Wang PL, Afari N, Jiang C, et al. Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development. Genome Biol 2015; 16: 126.

  • 10.

    Hall B, Limaye A, Kulkarni AB. Overview: generation of gene knockout mice. Curr Protoc Cell Biol 2009; 44: 19.12. 1-19.12.17.

  • 11.

    Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014; 157: 1262-1278.

  • 12.

    Koonin EV, Makarova KS. Origins and evolution of CRISPR-Cas systems. Philos Trans R Soc Lond B Biol Sci 2019; 374: 20180087.

  • 13.

    Eskandarian Boroujeni M. Genetically modified mice - Methods, applications and outlook. Sci J Kurdistan Univ Med Sci 2019; 24: 24-44.

  • 14.

    Zangala T. Isolation of genomic DNA from mouse tails. J Vis Exp 2007; 6: e246.

  • 15.

    Miyamoto T, Sengoku K, Hasuike S, Takuma N, Hayashi H, Yamashita T, Ishikawa M. Isolation and expression analysis of the human testis-specific gene, SPERGEN-1, a spermatogenic cell-specific gene-1. J Assist Reprod Genet 2003; 20: 101-104.

  • 16.

    Austin CP, Battey JF, Bradley A, Bucan M, Capecchi M, Collins FS. et al. The knockout mouse project. Nature Genet 2004; 36: 921.

  • 17.

    Sosa MG, De Gasperi R, Elder GA. Animal transgenesis: an overview. Brain Struct Funct 2010; 214: 91-109.

  • 18.

    Fujihara Y, Ikawa M. CRISPR/Cas9-based genome editing in mice by single plasmid injection, in Methods in enzymology. Methods Enzymol 2014; 546: 319-336.

  • 19.

    Nasseri S, Nikkho B, Parsa S, Ebadifar A, Soleimani F, Rahimi K, et al. Generation of Fam83h knockout mice by CRISPR/Cas9mediated gene engineering. J Cell Biochem 2019; 120: 11033-11043.

  • 20.

    Lee H, Kim JI, Park JS, Roh JI, Lee J, Kang BC, Lee HW. CRISPR/Cas9-mediated generation of a Plac8 knockout mouse model. Lab Anim Res 2018; 34: 279-287.