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

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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 β-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 bio-safety. These findings suggest that the SB transposon system is active and can be used for efficient gene transfer and insertional mutagenesis in tilapia.

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Acknowledgments

We are grateful to Drs. Qinjin Xu and I-Farn Lei for their careful reading and suggestions. We thank all other members in Qingdao tilapia seed multiplication farm (Qingdao, China) for their helps with the maintenance of experimental tilapia and all other members in Dr. Cui’s laboratory for helpful suggestions and technical assistance. This work was funded by grants from the National High-tech R&D (863) Program (#2007AA10Z164 to Z. Cui) and the National Natural Science Foundation of China (#31101892 to Y. Long).

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Authors and Affiliations

  1. The Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, 7 Donghu Rd, Wuhan, Hubei, 430072, People’s Republic of China

    Xiaozhen He, Jie Li, Yong Long, Guili Song, Zuoyan Zhu & Zongbin Cui

  2. Graduate University of the Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

    Xiaozhen He & Jie Li

  3. Qingdao Tilapia seed multiplication farm, Jiaozhou, 266317, People’s Republic of China

    Peiyong zhou & Qiuxiang Liu

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  1. Xiaozhen He
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  2. Jie Li
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  3. Yong Long
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  4. Guili Song
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  8. Zongbin Cui
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Correspondence to Zongbin Cui.

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Xiaozhen He and Jie Li contribute equally to this work and should be considered as co-first authors.

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Supplementary material 1 (DOC 58 kb)

11248_2013_9693_MOESM2_ESM.tif

Supplementary material Figure S1. The design and optimization of PCR primers. (A) Primers designed on the transgene are shown in the upper panel. Primer sets used for detection of transgenic tilapia and lengths (bp) of PCR products are shown in the lower panel. (B) The sensitivity and specificity of six primer sets were determined by PCR in a 25 μL volume containing 100 ng genomic DNA as template and transgenic plasmids (0, 1, 5, 10, 20, 50 or 100 copies). (TIFF 1606 kb)

11248_2013_9693_MOESM3_ESM.tif

Supplementary material 3 Figure S2. PCR screening of transgenic tilapia. (A) PCR of P0 fish with the primer set pXf/pYr. P: positive control using the transgenic plasmid as the template; WT: genomic DNA of wild type fish as the template; M: DNA marker; 1 ~ 45: genomic DNA samples of P0 tilapia. (B) PCR of F1 fish with the primer set pXf/pYr. P: positive control using the transgenic plasmid as the template; WT: genomic DNA of wild type fish as the template; M: DNA marker; 1’ ~ 219’: genomic DNA samples of F1 tilapia. (C) PCR of F2 fish with the primer set pXf/pYr. P: positive control using the transgenic plasmid as the template; WT: genomic DNA of wild type fish as the template; M: DNA marker; 1’’ ~ 91’’: genomic DNA samples of F2 tilapia. (TIFF 2581 kb)

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He, X., Li, J., Long, Y. et al. Gene transfer and mutagenesis mediated by Sleeping Beauty transposon in Nile tilapia (Oreochromis niloticus). Transgenic Res 22, 913–924 (2013). https://doi.org/10.1007/s11248-013-9693-8

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