Expression of Red Fluorescent Protein (RFP) in Transgenic Angelfish (Pterophyllum scalare Schultze, 1823)

Main Article Content

Vu Thanh Nguyen
Bui Hoang Loc
Vy Nguyen Hoang Thuy
Dinh Thi Thuy

Abstract

The freshwater angelfish (Pterophyllum scalare Schultze, 1823) is a common aquarium species. Similar to many other cichlids, angelfish provide parental care for their embryos and larvae. In contrast to zebrafish and medaka, it is difficult to raise angelfish eggs in the lab. Therefore, obtaining angelfish embryos for transgenic purposes will also be challenging. Our aim in this study was to employ microinjection techniques to develop transgenic angelfish expressing red fluorescent protein (RFP). Using the zebrafish myosin light chain 2 (mylz2) promoter, the expression patterns of the RFP founder transgenic angelfish were analyzed. An open loop of plasmid pDsred2-1 was cloned with the 1999-bp Mylz2 promoter fragment at the SacI and AgeI sites to generate the pMylz2-RFP transgenic construct. After angelfish laid their eggs on the substrate, the embryos were carefully collected with a plastic pipette in preparation for the microinjection procedure. A single-cell-stage angelfish embryo was microinjected with pMylz2-RFP. Sixteen of 524 pMylz2-RFP microinjected embryos survived to five days post fertilization (5 dpf), with twelve displaying red fluorescence. Only two RFP-positive larvae survived to adulthood. In adult angelfish, red LED illumination clearly displayed RFP expression in trunk muscles, indicating successful transmission of the transgene. The pMylz2-RFP transgene, however, was not passed on to offspring, indicating that it was not incorporated into the germline.

Article Details

How to Cite
Nguyen, V. T. ., Loc, B. H. ., Thuy, V. N. H. ., & Thuy, D. T. . (2022). Expression of Red Fluorescent Protein (RFP) in Transgenic Angelfish (Pterophyllum scalare Schultze, 1823). Journal of Fisheries and Environment, 46(3), 72–83. Retrieved from https://li01.tci-thaijo.org/index.php/JFE/article/view/257063
Section
Articles

References

Aivaz, A.N., A. Manica, P. Neuhaus and K.E. Ruckstuhl. 2020. Picky predators and odd prey: Colour and size matter in predator choice and zebrafish’s vulnerability-a refinement of the oddity effect. Ethology Ecology and Evolution 32: 135-147. DOI: 10.1080/03949370.2019.1680445.

Amsterdam, A., S. Lin and N. Hopkins. 1995. The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos. Developmental Biology 171: 123-129. DOI: 10.1006/dbio.1995.1265.

Bertola, L.D., E.B. Ott, S. Griepsma, F.J. Vonk and C.P. Bagowski. 2008. Developmental expression of the alpha-skeletal actin gene. BMC Evolutionary Biology 8: 166. DOI: 10.1186/1471-2148-8-166.

Çelik, I., P. Çelik, M. Gürkan and T. Sahin. 2014. Larval development of the freshwater angelfish Pterophyllum scalare (Teleostei: Cichlidae). Turkish Journal of Fisheries and Aquatic Sciences 14: 463-470. DOI: 10.4194/1303-2712-v14_4_03.

Chen, T.T., C.M. Lin, M.J. Chen, J.H. Lo, P.P. Chiou, H.Y. Gong, J.L. Wu, M.H.C. Chen and C. Yarish. 2015. Transgenic technology in marine organisms. In: Springer Handbook of Marine Biotechnology (ed. S.K. Kim), pp. 387-412. Springer, Berlin, Heidelberg, Germany.

Cho, Y.S., S.Y. Lee, D.S. Kim and Y.K. Nam. 2013. Characterization of stable fluorescent transgenic marine medaka (Oryzias dancena) lines carrying red fluorescent protein gene driven by myosin light chain 2 promoter. Transgenic Research 22: 849-59. DOI: 10.1007/s11248-012-9675-2.

Cho, Y.S., S.Y. Lee, Y.K. Kim, D.S. Kim and Y.K. Nam. 2011. Functional ability of cytoskeletal β-actin regulator to drive constitutive and ubiquitous expression of a fluorescent reporter throughout the life cycle of transgenic marine medaka Oryzias dancena. Transgenic Research 20: 1333-55. DOI: 10.1007/s11248-011-9501-2.

Chou, C.Y., L.S. Horng and H.J. Tsai. 2001. Uniform GFP-expression in transgenic medaka (Oryzias latipes) at the F0 generation. Transgenic Research 10: 303-315. DOI: 10.1023/a:1016671513425.

Debode, F., A. Marien, Q. Ledoux, E. Janssen, C. Ancion and G. Berben. 2020. Detection of ornamental transgenic fish by real-time PCR and fluorescence microscopy. Transgenic Research 29: 283-294. DOI: 10.1007/s11248-020-00197-9.

Dunham, R.A. and R.N. Winn. 2014. 11-Production of transgenic fish. In: Transgenic Animal Technology, 3rd ed. (ed. C.A.B.T.T.A.T Pinkert), pp. 305-334. Elsevier, London, UK.

Ge, J., Z. Dong, J. Li, Z. Xu, W. Song, J. Bao, D. Liang, J. Li, K. Li, W. Jia, M. Zhao, Y. Cai, J. Yang, J. Pan and Q. Zhao. 2012. Isolation of yellow catfish β-actin promoter and generation of transgenic yellow catfish expressing enhanced yellow fluorescent protein. Transgenic Research 21: 995-1004. DOI: 10.1007/s11248-012-9606-2.

Gong, Z., H. Wan, T.L. Tay, H. Wang, M. Chen and T. Yan. 2003. Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle. Biochemical and Biophysical Research Communications 308: 58-63. DOI: 10.1016/S0006-291X(03)01282-8.

Hamada, K., K. Tamaki, T. Sasado, Y. Watai, S. Kani, Y. Wakamatsu, K. Ozato, M. Kinoshita, R. Kohno, S. Takagi and M. Kimura. 1998. Usefulness of the medaka beta-actin promoter investigated using a mutant GFP reporter gene in transgenic medaka (Oryzias latipes). Molecular Marine Biology and Biotechnology 7: 173-180.

Jian, Q., M. Chen, J. Bai, P. Jiang, J. Fan, X. Ye and S. Xia. 2012. Generation and characterization of a stable red fluorescent transgenic Tanichthys albonubes line. African Journal of Biotechnology 11: 7756-7765. DOI: 10.5897/AJB11.3273.

Ju, B., S.W. Chong, J. He, X. Wang, Y. Xu, H. Wan, Y. Tong, T. Yan, V. Korzh and Z. Gong. 2003. Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the mylz2 promoter. Developmental Dynamics 227: 14-26. DOI: 10.1002/dvdy.10273.

Kinoshita, M. 2004. Transgenic medaka with brilliant fluorescence in skeletal muscle under normal light. Fisheries Science 70: 645-649. DOI: 10.1111/j.1444-2906.2004.00852.x.

Lee, S.Y., D.S. Kim, Y.K. Nam, S. Yoon, L. Dong, S. Kim and Y. Kwon. 2013. Molecular characterization of fast skeletal muscle-specific myosin light chain 2 gene (mlc2f) in marine medaka Oryzias dancena. Genes and Genomics 35: 289-303. DOI: 10.1007/s13258-013-0071-y.

Lin, Y.H., K.C. Peng, C.Y. Pan, Z.H. Wen and J.Y. Chen. 2014. Expression characterization and promoter activity analysis of the tilapia (Oreochromis niloticus) myosin light chain 3 promoter in skeletal muscle of fish. Transgenic Research 23: 125-134. DOI: 10.1007/s11248-013-9758-8.

Pan, X., H., Zhan and Z. Gong. 2008. Ornamental expression of red fluorescent protein in transgenic founders of white skirt tetra (Gymnocorymbus ternetzi). Marine Biotechnology 10: 497-501. DOI: 10.1007/s10126-008-9094-9.

Penman, D., A. Beeching, S. Penn and N. Maclean. 1990. Factors affecting survival and integration following microinjection of novel DNA into rainbow trout eggs. Aquaculture 85: 35-50. DOI: 10.1016/0044-8486(90)90005-8.

Slavík, O., P. Horký and M. Wackermannová. 2016. How does agonistic behaviour differ in albino and pigmented fish? PeerJ 4: e1937. DOI: 10.7717/peerj.1937.

Tanaka, M. and M. Kinoshita. 2001. Recent progress in the generation of transgenic medaka (Oryzias latipes). Zoological Science 18: 615-622. DOI: 10.2108/zsj.18.615.

Thilakarathne, K.G.D.D., G.N. Hirimuthugoda, P.H.T. Lakkana and S. Kumburegama. 2021. Embryonic and larval development in the freshwater angelfish (Pterophyllum scalare). Sri Lanka Journal of Aquatic Sciences 26: 25. DOI: 10.4038/sljas.v26i1.7586.

Thuy, V.N.H., T.M.N. Thanh, B.N. Quoc, H.N.T. Kieu, D. Van Nguyen, T.L.T. Thach and V.T. Nguyen. 2021. Generation of transgenic medaka Oryzias curvinotus (Nichols & Pope, 1927) carrying a cyan fluorescent protein gene driven by alpha actin promoter. Asian Fisheries Science 34: 56-62. DOI: 10.33997/j.afs.2021.34.1.006.

Vu, N.T., Y.S. Cho, S.Y. Lee, D.S. Kim and Y.K. Nam. 2014. A Cyan fluorescent protein gene (cfp)-transgenic marine medaka Oryzias dancena with potential ornamental applications. Fisheries and Aquatic Sciences 17: 479-486. DOI: 10.5657/FAS.2014.0479.

Wan, H., J. He, B. Ju, T. Yan, T.J. Lam and Z. Gong. 2002. Generation of two-color transgenic zebrafish using the green and red fluorescent protein reporter genes gfp and rfp. Marine Biotechnology 4: 146-154. DOI: 10.1007/s10126-001-0085-3.

Xu, Y., J. He, H.L. Tian, C.H. Chan, J. Liao, T. Yan, T.J. Lam and Z. Gong. 1999. Fast skeletal muscle-specific expression of a zebrafish myosin light chain 2 gene and characterization of its promoter by direct injection into skeletal muscle. DNA and Cell Biology 18: 85-95. DOI: 10.1089/104454999315655.

Zeng, Z., X. Liu, S. Seebah and Z. Gong. 2005. Faithful expression of living color reporter genes in transgenic medaka under two tissue-specific zebrafish promoters. Developmental Dynamics 234: 387-392. DOI: 10.1002/dvdy.20491.