Gene delivery to Nile tilapia cells for transgenesis and the role of PI3K-c2\u03b1 in angiogenesis

Fernanda Maria Policarpo Tonelli,Samyra Maria Dos Santos Nassif Lacerda,Luiz Renato De França,Rodrigo Ribeiro Resende,Marcela Santos Procópio,Breno Luiz Sales Lemos

doi:10.1038/srep44317

Fernanda Maria Policarpo Tonelli, Samyra Maria Dos Santos Nassif Lacerda + Show 4 more

Open Access

https://doi.org/10.1038/srep44317

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Abstract

Microinjection is commonly performed to achieve fish transgenesis; however, due to difficulties associated with this technique, new strategies are being developed. Here we evaluate the potential of lentiviral particles to genetically modify Nile tilapia cells to achieve transgenesis using three different approaches: spermatogonial stem cell (SSC) genetic modification and transplantation (SC), in vivo transduction of gametes (GT), and fertilised egg transduction (ET). The SC protocol using larvae generates animals with sustained production of modified sperm (80% of animals with 77% maximum sperm fluorescence [MSF]), but is a time-consuming protocol (sexual maturity in Nile tilapia is achieved at 6 months of age). GT is a faster technique, but the modified gamete production is temporary (70% of animals with 52% MSF). ET is an easier way to obtain mosaic transgenic animals compared to microinjection of eggs, but non-site-directed integration in the fish genome can be a problem. In this study, PI3Kc2α gene disruption impaired development during the embryo stage and caused premature death. The manipulator should choose a technique based on the time available for transgenic obtainment and if this generation is required to be continuous or not.

Highlights

Fish transgenesis has received attention since the early 1980s, when Mclean and Talwar genetically modified rainbow trout (Oncorhynchus mykiss) by microinjecting newly fertilised eggs[1]
The lentiviral particles were generated at a titre of 6.5 × 105 TU/mL determined in HeLa cells; they were used to genetically modify Nile tilapia cells to achieve transgenesis
The fish that developed from eggs exposed to lentiviral particles were mosaic animals (Fig. 1A)

Summary

Introduction

Fish transgenesis has received attention since the early 1980s, when Mclean and Talwar genetically modified rainbow trout (Oncorhynchus mykiss) by microinjecting newly fertilised eggs[1]. We analyse three strategies based on lentiviral particles as gene delivery vehicles: newly fertilised egg transduction, in vivo transduction of spermatozoa in adult fish testis, and in vitro genetic modification of spermatogonial stem cells (SSCs) followed by their transplantation into Nile tilapia larvae. Researchers have used these techniques to generate transgenic mice and rats. Lentiviruses can integrate in regions that encode gene products This integration can cause problems by disrupting some important genes, but it can be used as an important tool for studying gene function by observing the effects of insertional mutagenesis[11]

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