• the molecular structures, genetic regulations and function analyses of myf-5, troponin, cmlc, and rhodopsin
• using transgenic fish to study human diseases
• developing transgenic system and new species of aquacultural organism
• developing various body colors of ornamental fish

By using transgenic methods in vivo, we have sequenced the genes of zevrafish myf-5 1,2,3, cmlc2 4, and carp rhodopsin 5,6,7 that are responsible of controlling expressing specificities in tissues, as well as getting to know the proteins that bind them. We’ve also elaborated the roles that myf-5 1,8, pavalbumin 9, troponin 10 and intacalcin 11 genes might play in zebrafish embryonic development. Some useful model species like zebrafish and medaka have been used to develop transgenic species, based on regulating upstream sequences of myf-5, rhodopsin, troponin, and actin genes to generate reporter genes (like GFP and RFP). The steady transgenic species that we have in our laboratory now are zebrafish with green fluorescence either in heart 4,12,13, eye, or oval 14, and zebrafish as well as mesaka that have visible bright fluorescence under normal light 15,16. Besides, we also produce transgenic aquacultural animals that have the advantage to grow fast, like loach 17, abalone 18, and prawn 19,20. They are all obtained by using sperms as carriers successfully. These new transgenic species are of academic research and industrial application uses.

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1. Chen, Y. H., C. T. Liang and H. J. Tsai* 2002. Expression, purification and DNA-binding activity of tilapia muscle-specific transcription factor MyoD produced by Escherichia coli. Comp. Biochem. Physiol., 131B: 795-805. (SCI Journal)

2. Chen, Y. H. and H. J. Tsai* 2002. Treatment with Myf5-morpholino results in somite patterning and brain formation defects in zebrafish. Differentiation, 70: 447-456. (SCI Journal)

3. Ho, Y. L., Y. H. Shau,H. J.Tsai, L. C.Lin, P. J. Huang and F. J. Hsieh* 2002. Assessment of zebrafish cardiac performance using doppler echocardiography and power angiography. Ultrasound Med. Biol., 28: 1137-1143. (SCI Journal)

4. Wang, T. M., Y. H. Chen, C. F. Liu and H. J. Tsai* 2002. Functional analysis of the proximal promoter region of fish rhodopsin and Myf-5 genes using transgenesis. Marine Biotechnol., 4: 247-255. (SCI Journal)

5. Hsiao, C. D., W. Y. Tsai and H. J. Tsai* 2002. Isolation and expression of two zebrafish homologous of parvalbmin genes related to chicken CPV3 and mammalian oncomodulin. Mech. Dev., 119S:S161-S166. (SCI Journal)

6. Hsiao, C. D., W. Y. Tsai, L. S. Horng and H. J. Tsai* 2003. Molecular structure and developmental expression of three muscle-type of troponin T genes in zebrafish. Dev. Dynamics, 227:266-279. (SCI Journal)

7.  Huang, C. J., C. T. Tu, C. D. Hsiao, F. J. Hsieh and H. J. Tsai* 2003. Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish. Dev. Dynamics, 228:30-40. (SCI Journal)

8. Chen, Y. H., H. C. Lee, C. F. Liu, C. Y. Lin and H. J. Tsai* 2003. Novel regulatory sequence -82/-62 functions as a key element to drive the somite-specificity of zebrafish myf-5. Dev. Dynamics, 228:41-50. (SCI Journal)

9. Hsiao, C. D. and H. J. Tsai* 2003. Transgenic zebrafish with fluorescent germ cells: a useful tool to visualize germ cells proliferation and juvenile hermaphroditism in vivo. Dev. Biol., 262:313-323. (SCI Journal)

10.  Raya, A., C. M. Koth, D. Buscher, Y. Kawakami, T. Itoh, R. M. Raya, G. Sternik, H. J. Tsai,   C. Rodriguez-Esteban and J. C, Izpisua-Belmonte* 2003. Activation of Notch signaling pathway precedes heart regeneration in zebrafish. Proc. Natl. Acad. Sci. U.S.A.,100: 11889-11895. (SCI Journal)

11.  Shu, X., K. Cheng, N. Pate, F. Chen, E. Joseph, H. J. Tsai and J. Chen*. 2003. Na, K-ATPase is essential for embryonic heart development in the zebrafish. Development, 130:6165-6173. (SCI Journal)

12.  Hsiao, C. D., M. Ekker and H. J. Tsai* 2003. Skin-specific expression of ictacalcin, a homolog of the S100 genes during zebrafish embryogenesis.Dev. Dynamics, 228:745–750. (SCI Journal)

13.  Chu, S. W., T. M. Liu, C. K. Sun,* C. Y. Lin and H. J. Tsai. 2003. Real-time second- harmonic- generation microscopy based on a 2-GHz repetition rate Ti:sapphire Laser. Optics Express., 11:933-938. (SCI Journal)

14.  Chu, S. W., S. Y. Chen, T. H. Tsai, T. M. Liu, C.Y. Lin, H. J. Tsaiand C. -K. Sun.* 2003. In vivo developmental biology study using noninvasive multi-harmonic generation microscopy. Optics Express., 11:3093-3099. (SCI Journal)

15. Tsai, H. J. * 2003. Transgenic fish: research and application. J. Fish. Soc. Taiwan, 30:263-277 (invited review article)

16. Sun, C. K.,* S. -W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C. -Y. Lin and H. J. Tsai. 2004. Higher harmonic generation microscopy for developmental biology. J. Struct. Biol., 147: 19-30 (SCI Journal)

17. Lin, C. Y., Y. H. Chen, H. C. Lee and H. J. Tsai*. 2004. Novel cis-element in intron 1 represses somite expression of zebrafish myf-5. Gene, 334:63-72. (SCI Journal)

18. Huang, C. J., T. S. Jou, Y. L. Ho, W. H. Lee, Y. T. Jeng, F. J. Hsieh and H. J. Tsai*. 2005. Conditional expression of a myocardium-specific transgene in zebrafish transgenic lines. Dev. Dynamics, 233:1294-1303 (SCI Journal)

19. Wang, Y. H., Y. H. Chen, J. H. Lu and H. J. Tsai*. 2005. A 23-amino-acid residue spanning the basic domain targets zebrafish myogenic regulatory factor Myf-5 into nucleolus. DNA Cell Biol. 24: 651-660. (SCI Journal)

20. Chen, H. L., H. S. Yang, R. Huang and H. J. Tsai*. 2006. Transfer of a foreign gene to Japanese abalone (Haliotis diversicolor supertexta) by direct testis-injection. Aquaculture, 253:249-258. (SCI Journal)

21. Lee, H. C., H. Y. Huang, C. Y. Lin, Y. H. Chen and H. J. Tsai*. 2006. Foxd3 mediates zebrafish myf5 expression during early somitogenesis Dev. Biol. 290:359-372 (SCI journal)

22. Wang, Y. H., Y. H. Chen, Y. J. Lin and H. J. Tsai*. 2006. Spatiotemporal expression of zebrafish keratin 18 during early embryogenesis and the establishment of a keratin 18:RFP transgenic line. Mech. Dev.:Gene Expression Patterns 6:335-339. (SCI journal)

23. Tsai, T. H., C. Y. Lin, H. J. Tsai, S. Y. Chen, S. P. Tai, K. H. Lin and C. K. Sun*. 2006. Biomolecular imaging based on far-red fluorescent protein with a high two-photon excitation action cross section. Opt. Lett. 31: 930-932. (SCI Journal)

24. Wang, Y. H., Y. H. Chen, J. H. Lu, Y. J. Lin, M. Y. Chang and H. J. Tsai*. 2006. Epidermis-restricted expression of zebrafish cytokeratin II is controlled by a -141/+85 minimal promoter, and cassette -141/-111 is esential for driving the tissue-specificity. Differentiation 74:186-194. (SCI journal)

25. Wang, Y. H., Y. H. Chen, T. N. Wu and H. J. Tsai*. 2006. A keratin 18 transgenic zebrafish Tg(k18(2.9):RFP) treated with inorganic arsenite reveals visible overproliferation of epithelial cells. Toxicol. Lett. 163:191-197. (SCI journal)

26. Sato, M., H. J. Tsai and H. J. Yost*. 2006. Semaphorin3D regulates invasion of cardiac neural crest cells into the primary heart field. Dev. Biol. 298: 12-21. (SCI Journal)

27. Chun, C. Z., H. J. Tsai and T. T. Chen*. 2006. Trout Ea4- or Human Eb-Peptide of Pro-IGF-I disrupts heart, red blood cell, and vasculature development in zebrafish embryos. Mol. Reprod. Dev. 73:1112–1121. (SCI Journal)

28. Forouhar, A. S., M. Liebling, A. Hickerson, A. Nasiraei-Moghaddam, H. J. Tsai, J. R. Hove, S. E. Fraser, M. E. Dickinson and M. Gharib*. 2006. The embryonic vertebrate heart tube is a dynamic suction pump. Science 312: 751-753 (SCI Journal)

29. Sabaawy, H. E., M. Azuma, L. J. Embree, H. J. Tsai, M. F. Starost and D. D. Hickstein*. 2006. TEL-AML1 transgenic zebrafish model of precursor B cell acute lymphoblastic leukemia. Proc. Natl. Acad. Sci. USA 103: 15166-15171. (SCI Journal)

30. Lin, C. Y., R. F. Yunag, H. C. Lee, W. T. Chen, Y. H. Chen and H. J. Tsai*. 2006.Myogenic regulatory factors Myf5 and Myod function distinctly during craniofacial myogenesis of zebrafish. Dev. Biol. 299: 594-608. (SCI Journal)

31. Hu, C. Y., C. H. Yang, W. Y. Chen, C. J. Huang, H. Y. Huang, M. S. Chen and H. J. Tsai*. 2006. Egr1 gene knockdown affects embryonic ocular development in zebrafish. Mol. Vision 12: 1250-1258. (SCI Journal)

32. Chen, S. Y., C. Hsieh, S. Chu, C. Y. Lin, C. Ko, Y. Chen, H. J. Tsai, C. H. Hu and C. K. Sun*. 2006. Noninvasive harmonics optical microscopy for long-term observation of embryonic nervous system development in vivo. J. Biomed. Opt. 11: 054022-8. (SCI Journal)

33. Chen, Y. H., Y. H. Wang, M. Y. Chang, C. Y. Lin, C. W. Weng, M. Westerfield and H. J. Tsai*. 2007. Multiple upstream modules regulate zebrafish myf5 expression. BMC Dev. Biol. 7: 1-14 (SCI Journal)

34. Ho, Y. L., Y. H. Shau,L. C.Lin, P. J. Huang, F. J. Hsieh and H. J. Tsai*. 2007.In vivoassessment of cardiac morphology and function in heart-specific green fluorescent zebrafish. J. Formos. Med. Assoc. 106: 181-186.

35. H. C. Lee, J. N. Tsai, P. Y. Liao, W. Y. Tsai, K. Y. Lin, C. C. Chuang, C. K. Sun, W. C. Chang, H. J. Tsai.*. 2007. Glycogen synthase kinase 3alpha and 3beta have distinct functions during cariogenesis of zebrafish embryo. BMC Dev Biol. 7:93.

36. Holtzman, N. G., J. J. Schoenebeck, H. J. Tsai and D. Yelon*. 2007. Endocardium is necessary for cardiomyocyte movement during heart tube assembly. Development.134, 2379-86. (SCI journal)

37. Auman, H. J., H. Coleman, H. E. Riley, F. Olale, H. J. Tsai and D. Yelon*. 2007. Functional modulation of cardiac form through regionally confined cell shape changes. PLoS Biol. 5, e53. (SCI journal)

38. Wang, Y. H., C. K. Li, G. H. Le, H. J. Tsai * and Y. H. Chen*. 2008. Inactivation of the myogenic bHLH gene mrf4 in zebrafish leads to myofibril misalignment and motor axon growth disorganization in zebrafish embryos. Dev. Dynamics. 237, 1043-1050. (SCI journal)

39. Chen, Y. H.*, and H. J. Tsai *. 2008. Myogenic regulatory factors Myf5 and Mrf4 of fish: Current status and perspective. J. Fish. Biol. 73, 1-19. (reviewed and invited article)(SCI journal)

40. Chen, S. L., S. S. Li, R. Huang and H. J. Tsai *. 2008. Conditional production of a functional fish hormone in the transgenic line of Nannochloropsis oculata . J. Phycol. 44: 768-776. (SCI journal)

41. Li, S. S., and H. J. Tsai *. 2009. Transgenic microalgae as a non-antibiotic bactericide producer to defense the bacterial pathogen infection in fish digestive tract. Fish Shellfish Immunol. 26: 316-325. (SCI journal)

42. Tu, C. T., T. C. Yang and H. J. Tsai *. 2009. Nkx2.7 and Nkx2.5 function redundantly and are required for cardiac morphogenesis of zebrafish embryos. PLoS ONE. 4: e4249. (SCI journal)

43. Huang, H. Y., E. S. Dai, J. T. Liu, C. T. Tu, T. C. Yang and H. J. Tsai *. 2009. The embryonic expression patterns and the knockdown phenotypes of zebrafish ADP-ribosylation factor-like 6 interacting protein gene. Dev. Dynamics 238: 232-240. (SCI journal)

44. Fu, C. Y., H. C. Lee and H. J. Tsai* . 2009. The molecular structures and expression patterns of zebrafish tropoin Ⅰ genes. Gene. Expr. Patterns 9:348-56. (SCI journal)

45. Lin, C. Y., W. T. Chen, H. C. Lee, P. H. Yang, H. J. Yang and H. J. Tsai* . 2009. Transcription factor six1a plays function in craniofacial myogenesis of the zebrafish. Dev. Biol. 331:152-166. (SCI journal)

46. Hsu, R. J ., H. J . Yung and H. J. Tsai *. 2009. Labeled microRNA pull-down assay system: an experimental approach for high-throughput identification of microRNA-target mRNAs . Nucleic Acids Res. 37(10):77-87. (SCI journal)

48. Lee, H. C., W. A. Tseng, F. Y. Lo, T. M. Liu and H. J. Tsai*. 2009. Foxd5 mediates anterior-posterior polarity through upstream modulator fgf signaling during zebrafish s omitogenesis . Dev. Biol. 336:232-245. (SCI journal)

49. Ohn, J., H. J. Tsai and M. Liebling. 2009. Joint dynamic imaging of morphogenesis and function in the developing heart. Organognesis, 5:1-8. (SCI journal)

50. Lin, C. Y ., P. H . Yang, C. L . Kao, H. I. Huang and H. J. Tsai*. 2010. Transgenic zebrafish eggs containing bactericidal peptide is a novel food supplement enhancing resistance to pathogenic infection of fish. Fish Shellfish Immunol. 28: 419-427. (SCI journal)

51. Hsu, R. J., C. Y. Lin, H. S. Hoi, S. K. Zheng, C. C. Lin, H. J. Tsai*. 2010.Novel intronic microRNA represses zebrafish myf5 promoter activity through silencing dickkopf-3 gene. Nucleic Acids Res. 38, 4384-4393. (SCI journal)

52. Raeker, M. O .,  A. N. Bieniek, A. S. Ryan, H. J. Tsai ,  K. M. Zahn ,  M. W. Russell * . 2010. Targeted deletion of the zebrafish obscurin A RhoGEF domain affects heart, skeletal muscle and brain development. Dev. Biol. 337:432-443. (SCI journal)

53. Hsu RJ, C. C. Lin, Y. F. Su and H. J. Tsai * . 2011. D ickkopf-3-related gene regulates the expression of zebrafish myf5 gene through phosphorylated P 38a -dependent Smad4 activity. J Biol Chem . 286, 6855-6864. (SCI journal)

54. Chang SH, B. J. Lee, Y. D. Chen, Y. C. Lee and H. J. Tsai * . 2011. Development of transgenic zooplankton Artemia as a bioreactor to produce exogenous protein. Transgenic Res. (in press) (SCI journal)

55. Tu, C. T., T. C. Yang, H. Y. Huang and H. J. Tsai*. 2012. Zebrafish arl6ip1 is required for neural crest development during embryogenesis. PLoS One. 7, e32899. (IF:4.095; BIOLOGY 12/84)

56. Chen, Y. H., H. C. Lee, R. J. Hsu, T. Y. Chen, Y. K. Huang, H. C. Lo, S. C. Hu, H. J. Harn, J. R. Jeng, C. K. Sun, S. Z. Lin and H. J. Tsai*. 2012. The toxic effect of Amiodarone on valve formation in the developing heart of zebrafish embryos. Reprod Toxicol. 33, 233-244. (IF:3.266; TOXICOLOGY 20/83)

57. Fu, C. Y., Y. F. Su, M. H. Lee, G. D. Chang and H. J. Tsai * . 2012. Zebrafish DKK3a regulates the promoter activity of myf5 through interaction with membrane receptor Integrinα6b. J Biol Chem. 287: 40031-40042. ( IF: 4.651; BIOCHEMISTRY & MOLECULAR BIOLOGY 62/290 )

58. Lin CY, Chen JS, Loo MR, Hsiao CC, Chang WY, Tsai HJ*. 2013. MicroRNA-3906 regulates fast muscle differentiation through modulating the target gene homer-1b in zebrafish embryos. PLOS ONE, 8, e70187.

59. Lin CY, Lee HC, Chen HC, Hsieh CC and Tsai HJ*. 2013. Normal function of Myf5 during gastrulation is required for pharyngeal arch cartilage development in zebrafish embryos. Zebrafish, 10, 486-499.

60. Lin CY, Lee HJ, Fu CY, Ding YY, Chen JC, Lee MH, Huang, WJ and Tsai HJ*. 2013. miR-1 and miR-206 target different genes to have opposing roles during angiogenesis in zebrafish embryos. Nature Communications, 4, 2829.

61. Lee HC, Lu BN, Huang HL, Chu C, Li HP, Tsai HJ*. 2014. Transgenic line huORFZ is an effective living bioindicator for detecting environmental toxicants. PLoS ONE, 9:e90160.

62. Chiang CY, Chen YL, Tsai HJ*. 2014. Different visible colors and green fluorescence were obtained from the mutated purple chromoprotein isolated from sea anemone. Marine Biotechnology, 16:436-446.

63. Lee HC, Lin YZ, Lai YT, Huang WJ, Hu JR, Tsai JN, Tsai HJ*. 2014. Glycogen synthase kinase 3 beta in somites plays a role during the angiogenesis of zebrafish embryos. FEBS Journal, 281:4367-83.

64. Shih CH, Chen HY, Lee HC, Tsai HJ*. 2015. Purple Chromoprotein Gene Serves as a New Selection Marker for Transgenesis of the Microalga Nannochloropsis oculata. PlosONE, 10:e0120780.

65. Chiang CY, Lee CC, Lo SY, Wang HJ, Tsai HJ*. 2015. Chromophore deprotonation state alters the optical properties of blue chromoprotein. PlosONE, 10: e0134108.

66. Lee HC, Su MY, Lo HC, Wu CC, Lo DM, Chao TY, Tsai HJ, Dai MS. 2015. Cancer metastasis and EGFR signaling is suppressed by Amiodaroneinduced Versican-V2. Oncotarget, 6:42976-42987.

67. Lee HC, Lo HC, Lo DM, Su MY, Hu JR, Wu CC, Chang SH, Dai MS, Tsai CT, Tsai HJ*. 2015. Amiodarone Induces Overexpression of Similar to Versican b to Repress the EGFR/Gsk3b/Snail Signaling Axis during Cardiac Valve Formation of Zebrafish Embryos. Plos ONE, 10:e0144751.

68. Fu CY, Wang PC, Tsai HJ*. 2016. Competitive binding between Seryl-tRNA synthetase/YY1 complex and NFKB1 at the distal segment results in differential regulation of human vegfa promoter activity during angiogenesis. Nucleic Acids Research, 45:2423–2437.

69. Chiang CY, Lin CY, Chen YT, Tsai HJ*. 2016. Blue Fluorescent Protein Derived from the Mutated Purple Chromoprotein Isolated from the Sea Anemone Stichodactyla haddoni. Protein Engineering, Design & Selection, 29:523-530.

70. Lin CY, Chiang CY, Tsai HJ*. 2016. Zebrafish and Medaka: new model organisms for modern biomedical research. J Biomed Sci, 23:19.

71. Zeng CW, Kamei Y, Wang CT, Tsai HJ*. 2016. Subtypes of hypoxiaresponsive cells differentiate into neurons in spinal cord of zebrafish embryos after hypoxic stress. Biology of the cell, 108:357-377.

72. Lin CY, He JY, Loo MR, Chang WY, Zeng CW, Tsai HJ*. 2017. microRNA-206 mediates boundary formation in newly forming somites through an Rtn4a/Cxcr4a/Thbs3a pathway. Open Biology, 7: 170009.

73. Lee HC, Fu CY, Zeng CW, Tsai HJ*. 2017. Embryonic expression patterns of Eukaryotic EndoU ribonuclease family gene endouC in zebrafish. Gene expression patterns, 26:66-70.

74. Lin CY, Lee HC, Wu JH, Tsai HJ*. 2018. Short fish-origin DNA elements served as flanking sequences in a knockdown cloning vector enabling the generation of a functional siRNA molecule in mammalian cells and fish embryos. Biochemical and Biophysical Research Communications, 505, 850-857.

75. Lin CY, Zhang PH, Chen YJ, Wu CL, Tsai HJ*. 2019. Conditional overexpression of rtn4al in muscle of adult zebrafish displays defects similar to human amyotrophic lateral sclerosis. Marine biotechnology, 21, 52-64.

76. Chang HY, Ko TP, Chang YC, Huang KF, Lin CY, Chou HY, Chiang CY, Tsai HJ*. 2019. Crystal structure of the blue fluorescent protein with a Leu-Leu-Gly tri-peptide chromophore derived from the purple chromoprotein of Stichodactyla haddoni. International Journal of Biological Macromolecules, 130, 675-684.

77. Lin CY, Wu CL, Lee KZ, Chen YJ, Zhang PH, Chang CY, Harn HJ, Lin SZ, Tsai HJ*.? 2019. Extracellular Pgk1 enhances neurite outgrowth of motoneurons through Nogo66/NgR- independent targeting of NogoA. eLIFE, 8:e49175.

78. Lee BC, Hung CW , Lin CY, Shih CH, Tsai HJ*. 2019. Oral administration of transgenic biosafe microorganism containing antimicrobial peptide enhances the survival of tilapia fry infected bacterial pathogen. Fish and Shellfish Immunology, 95:606-616.

79. Zeng CW, Sheu JC, Tsai HJ*. 2020. A new member of the forkhead box protein family in zebrafish: Domain composition, phylogenetic implication and embryonic expression pattern. Gene Expression Pattern, 35: 119093.