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.  

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