Gene transfer and mutagenesis mediated by Sleeping Beauty transposon in Nile tilapia (Oreochromis niloticus)

Abstract

The success of gene transfer has been demonstrated in many of vertebrate species, whereas the efficiency of producing transgenic animals remains pretty low due to the random integration of foreign genes into a recipient genome. The Sleeping Beauty (SB) transposon is able to improve the efficiency of gene transfer in zebrafish and mouse, but its activity in tilapia (Oreochromis niloticus) has yet to be characterized. Herein, we demonstrate the potential of using the SB transposon system as an effective tool for gene transfer and insertional mutagenesis in tilapia. A transgenic construct pT2/tiHsp70-SB11 was generated by subcloning the promoter of tilapia heat shock protein 70 (tiHsp70) gene, the SB11 transposase gene and the carp b-actin gene polyadenylation signal into the second generation of SB transposon. Transgenic tilapia was produced by microinjection of this construct with in vitro synthesized capped SB11 mRNA. SB11 transposon was detected in 28.89 % of founders, 12.9 % of F1 and 43.75 % of F2. Analysis of genomic sequences flanking integrated transposons indicates that this transgenic tilapia line carries two copies of SB transposon, which landed into two different endogenous genes. Induced expression of SB11 gene after heat shock was detected using reverse transcription PCR in F2 transgenic individuals. In addition, the Cre/loxP system was introduced to delete the SB11 cassette for stabilization of gene interruption and biosafety. These findings suggest that the SB transposon system is active and can be used for efficient gene transfer and insertional mutagenesis in tilapia.

Generation of Knockout and Transgenic Zebrafish to Characterize Abcc4 Functions in Detoxification and Efflux of Lead

“…Total genomic DNA from adult fish tails was isolated according to our previous study [51]. Three PCR primers were designed according to the coding sequences of the carp β-actin promoter, abcc4 CDS and Flag tag (Table 2).…”

A Review of Genetic Advances Related to Sex Control and Manipulation in Tilapia

“…Most transgenic research has been performed on the F0 generation (Kaneko et al ; Li et al ). A few successful cases of germline transmission of the transgene were focused on transposon mediated or β‐actin promoter‐driven EGFP and CMV promoter‐driven target gene expression (He et al ; Farlora et al ; Li et al ). As yet transgenic tilapia are not used in commercial production.…”

Sleeping Beautytransposition: from biology to applications

“…SB was successfully used as a delivery vehicle for germline transgenesis in a variety of vertebrate species like in fish, including zebrafish (Danio rerio) (Balciunas et al, 2004;Davidson et al, 2003), Nile tilapia (Oreochromis niloticus) (He et al, 2013), medaka (Oryzias latipes) (Grabher et al, 2003), in the amphibian Xenopus laevis (Sinzelle et al, 2006), in rodents (Garcia Diaz et al, 2016;Garrels et al, 2016c;Ivics et al, 2014c), rabbits (Ivics et al, 2014b), swine (Garrels et al, 2011;Ivics et al, 2014a) and cattle (Alessio et al, 2016;Garrels et al, 2016b;Yum et al, 2016).…”