Efficient Gene Transfer and Gene Editing in Sterlet (Acipenser ruthenus).

Ji Chen,Tian Dong,Wei Hu,Hongxia Hu,Zuoyan Zhu,Zhaohui Tian,Hua Zhu,Ying Dong,Wei Wang

doi:10.3389/fgene.2018.00117

Abstract

The sturgeon (Acipenseriformes) is an important farmed species because of its economical value. However, neither gene transfer nor gene editing techniques have been established in sturgeon for molecular breeding and gene functional study until now. In this study, we accomplished gene transfer and gene editing in sterlet (Acipenser ruthenus), which has the shortest sexual maturation period of sturgeons. The plasmid encoding enhanced green fluorescent protein (EGFP) was transferred into the embryos of sterlet at injection concentration of 100 ng/μL, under which condition high survival rate and gene transfer rate could be achieved. Subsequently, exogenous EGFP was efficiently disrupted by transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease/guide RNA (gRNA), with injection concentrations of 300 ng/μL TALENs, or 100 ng/μL Cas9 nuclease and 30 ng/μL gRNA, respectively, under which condition high survival rate and gene mutation rate could be achieved. Finally, the endogenous gene no tail in sterlet was successfully mutated by Cas9 nuclease/gRNA. We observed the CRISPR-induced no tail mutation, at a high efficiency with the mutant P0 embryos displaying the expected phenotype of bent spine and twisted tail.

Highlights

Since the first batch of transgenic fish was produced, more than 35 fish species have been modified using exogenous genes, which included commercial aquaculture fish such as common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idellus), Nile tilapia (Oreochromis niloticus), channel catfish (Ietalurus punetaus), rainbow trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch), Atlantic salmon (Salmo salar), and mud loach (Misgurnus mizolepis) (Hu and Zhu, 2010), as well as laboratory model fish such as zebrafish (Danio rerio) and medaka (Oryzias latipes)
Based on green fluorescence observed in embryos, the enhanced green fluorescent protein (EGFP) transfer rate increased with injected plasmid concentration
At 8 dpf, there were dots of fluorescence in fry bodies, which represented the foreign EGFP DNA unit that had been integrated into the cell genome (Figure 2B)

Summary

Introduction

Since the first batch of transgenic fish was produced, more than 35 fish species have been modified using exogenous genes, which included commercial aquaculture fish such as common carp (Cyprinus carpio), grass carp (Ctenopharyngodon idellus), Nile tilapia (Oreochromis niloticus), channel catfish (Ietalurus punetaus), rainbow trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch), Atlantic salmon (Salmo salar), and mud loach (Misgurnus mizolepis) (Hu and Zhu, 2010), as well as laboratory model fish such as zebrafish (Danio rerio) and medaka (Oryzias latipes). Until now, targeted gene editing has been reported in zebrafish, medaka, Nile tilapia, rainbow trout, common carp, channel catfish, rohu (Labeo rohita), Chinese tongue sole (Cynoglossus semilaevis), rice field eel (Monopterus albus), and so on (Doyon et al, 2008; Ansai et al, 2014; Li et al, 2014; Yano et al, 2014; Chakrapani et al, 2016; Qin et al, 2016; Zhong et al, 2016; Cui et al, 2017; Feng et al, 2017)

Methods

Results

Conclusion