本實驗室研究吳郭魚腦部基因之表現，並建立腦部表現序列
標記 (expressed sequence tags, ESTs) 資料庫，從中取得溫度感受
性神經發育相關基因 inhibitor of DNA binding/ differentiation
protein 2 (Id2) 序列進行生物資訊，包含開放閱讀區 (open reading
frame, ORF) 部分序列738bp，ORF為411bp，轉譯之蛋白質由 137
個胺基酸組成，與其他物種進行相似度比較之結果接近，亦繪製
16 個物種的親緣樹狀圖 (Neighbor Phylogenetic Tree)。本研究藉
由 6 個 His 作為標記，將 Id2 開放閱讀區建構於原核生物
(Escherichia coli) 表現系統中，再利用親和性管柱將 Id2 蛋白質
成功分離純化，作為後續之研究；並建構真核生物表現系統 (老
鼠神經瘤細胞, Neuro-2a)，將 Id2 建構在綠色螢光蛋白 (green
fluorescent protein, GFP) 表現載體上，轉染至 Neuro-2a 中，使
用螢光顯微鏡觀察以瞭解 Id2 在細胞的分布位置。

Expressed sequence tags (ESTs) are derived from the developing
tilapia brain was established in our lab. There are 9 transcripts were
identified as thermosensitive/Neural development-related gene. The
effects of different temperatures on the ontogenetic expression of
these thermosensitive/Neural development-related gene during the
critical period of brain sexual differentiation were investigated in the
present study. The ontogenetic expression of inhibitor of DNA
binding/differentiation protein 2 (Id2), thermosensitive/Neural
development-related gene, were enhanced by both lower (20℃) and
higher (32℃) temperatures before 10 days post-hatching.
In this study, bioinformatics were searched for Id2, which is a
gene with 738 bp of patial cDNA sequence, open reading frame
(ORF) is 411bp, and deduced 137 amino acids of protein sequence.
The protein of Id2 was expressed in a prokaryotic system, BL21
(Escherichia coli) and purified with Ni-NTA affinity chromatography.
Also, the ORF of Id2 was cloned into pEGFP vector, and
plasmid (pEGFP-Id2) was transfected into the eukaryotic system,
mouse neuroblastoma cell (Neuro-2a cell). The distribution of Id2
expressed in the Neuro-2a cell was identified by fluorescence
microscopy.

中文摘要…………………………………………………………………I
英文摘要 ……………………………………………………………… II
目錄 ……………………………………………………………………III
附圖、表之目錄…………………………………………………………IV
附錄之目錄 …………………………………………………………… V
前言………………………………………………………………………1
實驗材料與方法…………………………………………………………4
結果 ……………………………………………………………………31
討論 ……………………………………………………………………35
參考文獻 ………………………………………………………………38
附圖、表 ……………………………………………………………… 43
附錄 ……………………………………………………………………54

Brännvall K, Korhonen L, Lindholm D. (2002) Estrogen-receptordependent
regulation of neural stem cell proliferation and differentiation.
Mol Cell Neurosci. 21(3):512-520.
Bull JJ, Vogt RC. (1981). Temperature-sensitive periods of sex determination
in Emydid turtles. J Exp Zool. 218(3):435-440.
Campos-Ramos R, Harvey SC, Masabanda JS, Carrasco LAP, Griffin DK,
McAndrew BJ, Bromage NR, Penmanl DJ (2001). Identification of
putative sex chromosomes in the blue tilapia, Oreochromis aureus,
through synaptonemal complex and FISH analysis. Genetica.
111:143-153.
Carrer HF, Cambiasso MJ. (2002) Sexual differentiation of the brain:
genes, estrogen, and neurotrophic factors. Cell Mol Neurobiol.
22(5-6):479- 500.
Claro F, Del Abril A, Segovia S, Guillamon A. (1990). SBR: a computer
program to record and analyse sexual behavior in rodents. Physiol
Behav. 48(3):489-493.
Cordero ME, Valenzuela CY, Torres R, Rodriguez A. (2000). Sexual
dimorphism in number and proportion of neurons in the human
median raphe nucleus. Brain Res Dev Brain Res. 30;124(1-2):43-52.
del Abril A, Segovia S, Guillamón A. (1987). The bed nucleus of the stria
terminalis in the rat: regional sex differences controlled by gonadal
steroids early after birth. Brain. Res. 429(2):295-300.
D'Cotta H, Fostier A, Guiguen Y, Govoroun M, Baroiller JF. (2001a).
Search for genes involved in the temperature-induced gonadal sex
differentiation in the tilapia, Oreochromis niloticus. J Exp Zool.
290(6): 574-585.
D'Cotta H, Fostier A, Guiguen Y, Govoroun M, Baroiller JF. (2001b).
Aromatase plays a key role during normal and temperature-induced
sex differentiation of tilapia Oreochromis niloticus.Mol Reprod Dev.
59(3): 265-276.
Dournon C, Durand D, Demassieux C, Lesimple M. (1990). Differential
germ cell proliferation in the salamander pleurodeles waltl: controls
by sexual genotype and by thermal epigenetic factor before
differentiation of sexual phenotype of gonads. Int J Dev Biol.
34(3):365-375.
Gorski RA, Gordon JH, Shryne JE, Southam AM. (1978). Evidence for a
morphological sex differce within the medial preoptic area of the rat
brain. Brain. Res. 148(2):333-346.
Kocher TD, Lee WJ, Sobolewska H, Penman D, McAndrew B. (1998). A
genetic linkage map of a cichlid fish, the tilapia (Oreochromis
niloticus). Genetics. 148:1225–1232.
Kwon JY, McAndrew BJ, and Penman DJ. (2001). Cloning of brain
aromatase gene and expression of brain and ovarian aromatase genes
during sexual differentiation in genetic male and female Nile tilapia
Oreochromis niloticus. Mol. Reprod. Dev. 59:359-370.
Nishiyama H, Itoh K, Kaneko Y, Kishishita M, Yoshida O, Fujita J.
(1997). A glycine-rich RNA-binding protein mediating
cold-inducible suppression of mammalian cell growth. J Cell Biol.
137(4): 899-908.
Nopoulos P, Flaum M, O’Leary D, Andreasen NC. (2000). Sexual
dimorphism in the human brain: evaluation of tissue volume, tissue
composition and surface anatomy using magnetic resonance
imaging. Psychiatry Research. 98: 1-13.
Handa RJ, Hines M, Schoonmaker JN, Shryne JE, Gorski RA. (1986).
Evidence that serotonin is involved in the sexually dimorphic
development of the preoptic area in the rat brain. Devl. Brain. Res.
30: 278-282.
Segovia S, Guillamón A. (1993). Sexual dimorphism in the vomeronasal
pathway and sex differences in reproductive behaviors. Brain Res
Rev. 18(1):51-74.
Scobey MJ, Fix CA, Walker WH. (2004) The Id2 transcriptional
repressor is induced by follicle-stimulating hormone and cAMP. J
Biol Chen. 279(16):16064-16070.
Shiue YL, Wang LH, Chao TY, Lin CH, Tsai CL. (2004) EST-based
identification of genes expressed in the hypothalamus of adult tilapia,
Oreochromis mossambicus. Biochem Biophys Res Commun.
2;316(2): 523-7.
Swaab DF. (1995). Development of the human hypothalamus.
Neurochem. Res. 20(5):509-19.
Tajima K, Terai S, Takami T, Kawaguchi K, Okita K, Sakaida I. (2007).
Importance of inhibitor of DNA binding/ differentiation 2 in hepatic
stellate cell differentiation and proliferation. Hepatol Res.
37(8):647-655.
Toran-Allerand CD. (2004). Estrogen and the brain: beyond ER-alpha
and ER-beta. Exp Gerontol. 39(11-12):1579-1586.
Tsai CL, Wang LH, Shiue YL, Chao TY (2007) Influence of temperature
on the ontogenetic expression of neural development-related genes
from developing Tilapia brain expressed sequence tags. Mar
Biotechnol (NY). 9(2):243-261.
Tsai CL, Chang SL, Wang LH, Chao TY. (2003). Temperature influences
the ontogenetic expression of aromatase and estrogen receptor
mRNA in the developing tilapia (Oreochromis mossambicus) brain.
J Neuro- endocrinol. 15(1): 97-102.
T s a i CL , Wa n g LH, F a n g LS ( 2 0 0 1 ) . Es t r a d i o l a n d
parachlorophenylanine downregulate the expression of brain
aromatase and estrogen receptor a mRNA during the critical period
of feminizat ion in t i lapia, Oreochromi s mos sambicus .
Neuroendocrinology. 74, 325-334.
Tsai CL, Wang LH, Chang CF, Kao CC. (2000). Effects of gonadal
steroids on brain serotonergic and aromatase activity during the
critical period of sexual differentiation in tilapia, Oreochromis
mossambicus. J Neuro- endocrinol. 12(9): 894-8.
Uochi T, Asashima M. (1998). XCIRP (Xenopus homolog of
cold-inducible RNA-binding protein) is expressed transiently in
developing pronephros and neural tissue. Gene. 211(2): 245-50.
Wang LH, Tsai CL. (2006). Influence of temperature and gonadal
steroids on the ontogenetic expression of brain serotonin 1A and 1D
receptors during the critical period of sexual differentiation in tilapia,
Oreochromis mossambicus. Comp Biochem Physiol B Biochem Mol
Biol. 143(1):116-125.
Wang LH, Tsai CL. (2000a). Effects of temperature on the early
development of the central nervous system in tilapia, Oreochromis
mossambicus. Neurosci Lett. 285(2): 95-98.
Wang LH, Tsai CL. (2000b). Effects of temperature on the deformity and
sex differentiation of tilapia, Oreochromis mossambicus. J Exp Zool.
286(5): 534-537.
White AP, Collinson SK, Banser PA, Gibson DL, Paetzel M, Strynadka
NC, Kay WW. (2001). Structure and characterization of AgfB from
Salmonella enteritidis thin aggregative fimbriae. J Mol Biol.
24;311(4):735-749.
Zenke M, Hieronymus T. (2006). Molecular switches and developmental
potential of adult stem cells. Ernst Schering Res Found Workshop.
(60):69-79.