BIO 313 Zebra Fish Embryo.docx

Use the knockout technique to knock the gene out and

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use the knockout technique to knock the gene out and see how it effects the development of the embryo. This is important in a larger context because gene expression is important developmentally and can lead to bigger scientific breakthroughs. 8
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Figure 1. Gel electrophoresis of Pax2a and 2b with 1kb ladder scale. bp 1 2 3 4 5 6 7 8 9 10 9
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Figure 2. Gel electrophoresis of Pax1a and 1b with 1kb ladder scale. bp 1 2 3 4 5 6 7 8 9 10 10
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Table 1. Table containing expected and observed PCR product size for pax2a/2b and pax1a/1b 11
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Well Number PCR Reaction Expected product size Observed product size 1 27 hpf RNA plus -actin primers 114 500-250bp 1 27 hpf RNA plus -actin primers 114 500bp 2 75 hpf rna plus -actin primers 114 500-250bp 2 75 hpf rna plus -actin primers 114 none 3 no rna plus -actin primers none none 3 no rna plus -actin primers none none 4 27 hpf RNA plus pax2a primer 186 500-750bp 4 27 hpf RNA plus pax1a primer 186 none 5 75 hpf RNA plus pax2a primer 186 500-750bp 5 75 hpf RNA plus pax1a primer 186 none 6 No RNA plus pax2a primer None none 6 No RNA plus pax1a primer none none 7 27 hpf RNA plus pax2b primer 179 250bp 7 27 hpf RNA plus pax1b primer 179 none 8 75hpf RNA plus pax2b primer 179 250-500bp 8 75hpf RNA plus pax1b primer 179 none 9 No RNA plus pax2b primer none none 9 No RNA plus pax1b primer none none 10 Ladder n/a n/a
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References Brand, M., Heisenberg, C. P., Jiang, Y. J., Beuchle, D., Lun, K., Furutani-Seiki, M., Granato, M., Haffter, P., Hammerschmidt, M., Kane, D. et al. (1996). Mutations in zebrafish affecting the formation of the boundary between midbrain and hindbrain . Development 123,179 -190 Ebensperger C, Wilting J, Brand-Saberi B, Mizutani Y, Christ B, Bailing R, Koseki H: Pax- l, a regulator of sclerotome development is induced by notochord and floor plate signals in avian embryos. Anat Embryo /1995, 191:297-310. Garner, M., & Revzin, A. (1981). A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: Application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Research , 9(13), 3047-60. Jun Inoue, Yukuto Sato, Robert Sinclair, Katsumi Tsukamoto, & Mutsumi Nishida. (2015). Rapid genome reshaping by multiple-gene loss after whole-genome duplication in teleost fish suggested by mathematical modeling. Proceedings of the National Academy of Sciences , 112(48), 14918. Macdonald R, Wilson SW: Pax proteins and eye development . Curr Opin Neurobiol 1996, 6:49-56. Wolf Reik. (2007). Stability and flexibility of epigenetic gene regulation in mammalian development. Nature , 447(7143), 425-432. Zhiyuan, G., & Korzh, Vladimir. (2004). Fish development and genetics the zebrafish and medaka models (Molecular aspects of fish and marine biology ; v. 2). New Jersey: World Scientific Pub. 13
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