Generation of Global Spata19 Knockout Mouse Using Crispr/Cas9 Nickase Technology



Zargar M¹ ; Jamshidizad A² ; Rahimtayefeh A² ; Hashemi E² ; Najafi A¹ ; Shamsara M² ; Modarressi MH¹ Show Affiliations
Source: Koomesh Published:2020

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. © 2020, Semnan University of Medical Sciences. All rights reserved.