TITLE:
Construction of Zebrafish Mutants of CNTF Gene Using CRISPR/Cas9 Genome Editing Technology
AUTHORS:
Lihan Xu, Rong He, Feihao Shen, Keyun Wang, Yingjia Wang, Linyi Hu, Meihua Ye, Yan Zhang, Yuying Wang
KEYWORDS:
Environment Pollution, Ultraviolet Stabilizer, Parkinson’s Disease, Ciliary Neurotrophic Factor, Zebrafish, CRISPR/Cas9
JOURNAL NAME:
Journal of Biosciences and Medicines,
Vol.11 No.11,
November
23,
2023
ABSTRACT: Background: The prevalence of Parkinson’s disease (PD), a chronic and progressive neurodegenerative disorder, is projected to increase twofold by 2030. Leucine-rich repeat kinase 2 (LRRK2) is the most commonly observed gene in both familial and sporadic PD cases. Notably, there is a substantial augmentation in motor activity during both larval and adult stages of zebrafish lacking the lrrk2 gene. Nevertheless, the precise genetic abnormalities accountable for eliciting these phenotypes in zebrafish are yet to be elucidated. Methods: Real-time polymerase chain reaction (PCR) was conducted on zebrafish larvae at 6 days post fertilization (dpf) belonging to both the wild-type and lrk2(-/-) groups. Guide RNA was designed and subsequently employed in the PCR process. Electrophoresis was performed to facilitate identification. Results: The expression of CNTF mRNA was significantly diminished in lrrk2(-/-), in comparison to the wildtype zebrafish larvae. This finding implies that CNTF may have crucial implications in the regulated functioning of lrrk2, which is widely acknowledged as the predominant genetic factor contributing to hereditary PD. The primers for CNTF DNA were meticulously designed, and the electrophoresis results of the PCR product were subsequently presented. The wild type zebrafish embryos were meticulously prepared for micro-injection, and the resulting efficiency identification displayed the presence of the mutant PCR product, which exhibited the presence of several debris. Conclusions: The present study demonstrates the successful generation of CNTF mutant zebrafish using the CRISPR/Cas9 genome editing technique. Further investigations are necessary to deepen our understanding of the exogenous CNTF gene’s functionality.