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表一、PCR 反應所使用的 primer 序列 Delet
domain
Primer 名稱 sequence Site* PCR product
(bp)
5’ctagctagcatgcacctgcgtgtcctccgctg 3’
（包含 NheI 切位）
208~
227
1733
BC030547-F2 5’ctagctagcatgggccccctctcggcgc3’
（包含 NheI 切位）
1~
19 BC030547
delWD1-R12
gtctggcacacagcaggctg 802~
813
ctgctgtgtgccagacgggc 976~
987
965
BC030547-F2 5’ctagctagcatgggccccctctcggcgc3’
（包含 NheI 切位）
BC030547-F2 5’ctagctagcatgggccccctctcggcgc3’
（包含 NheI 切位）
BC030547 ccacggtggtcagcaacatcc 1744
197
delWD3-F14 ~ 1756
*NCBI accession no：BC030547
圖一、睪丸組織剖面圖。哺乳類動物之精子發育過程發生在睪丸組 織，睪丸是由許多曲精小管( seminiferous tubule ) 所組成，其內有精 原細胞 ( spermatogonia ) 及Sertoli cell，在曲精小管之間有Leydig cell。在整個細胞分化的過程中，精母細胞 ( spermatocytes ) 會慢慢 由曲精小管 ( seminiferous tubule ) 的外圍往中央的管腔 ( lumen ) 移動，而漸漸形成成熟的精子。
圖二、哺乳類動物之精子發育過程 ( Spermatogenesis )，分為三個階 段：(一)、精原細胞期 ( spermatogonia phase or spermatocytogenesis )；
(二)、減數分裂期 ( meiotic phase or meiosis )；(三)、精子形成期 ( spermatid phase or spermiogenesis ) ［19］。
圖三、造精功能障礙的睪丸組織病理切片染色。依造精功能受損的程 度可區分為四種不同的類型：a、正常造精功能的睪丸組織（Normal spermatogenesis, NR）； b、缺乏生殖細胞的睪丸組織（Sertoli cell-only syndrome, SCOS）；c、造精功能停滯的睪丸組織（Maturation arrest, MA）；d、造精功能低下的睪丸組織（Hypospermatogenesis, HS）。
SC：Sertoli cells、Spg：spermatogonia、Spc：spermatocyte 及Spd：
spermatid［35］。
圖四、本研究這次所使用的載體及 NheI、BamHI 切位的位置
圖五、LRR 和 3WD domain 所對應的 primer 方式
圖六、構築完成的pHaloTag-pHT2-LRWD1-del LRR 載體
圖七、構築完成的pHaloTag-pHT2-LRWD1-del WD1 載體
圖八、構築完成的pHaloTag-pHT2-LRWD1-del WD2 載體
圖九、構築完成的pHaloTag-pHT2-LRWD1-del WD3 載體
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
kb
0.3 0.2
0.1 3.0 2.0 1.5 1.0 0.9 0.8 0.7 0.6 0.5 0.4
圖十、構築完成的載體使用限制酶確認之電泳圖，每個 Land 的內容 列於表二。
表二、此表是圖十中每個 Land 的內容，每個構築完成的載體都使用 兩組限制酶做確認是否有確實剔除目標 domain。
M：100 bp ladder DNA marker
Land 1 2 3 4 5 6 7 8
595,1084 595,961 239,241, 396,1058,
Fluorescence DAPI Merge
wild
LRR
WD1
WD2
WD3
圖十一、免疫螢光分析（immunofluorescence assay, IFA）看 LRWD1 和其他剔除目標domain 表現細胞的位置。左排的文字所標明的 wild 是指沒有剔除任何domain，而 LRR、WD1、WD2、WD3 指的是剔
除的目標domain。從圖中看到的亮點，不能確認是本研究送入的質 體產生的LRWD1，有可能是 GC-2 cell 自己產生的 LRWD1，實驗中 使用的一級抗體是anti-LWRD1，所以也會與 GC-2 cell 本身生產 LRWD1 蛋白質作用。
170
130
100
70
55
Marker wild LRR WD1 WD2 WD3 kDa
圖十二、西方墨點法（Western blot）觀察 delete form 與 wild 大小差 異結果圖。上排的文字所標明的wild 是指沒有剔除任何 domain，而 LRR、WD1、WD2、WD3 指的是剔除的目標 domain。在 70kDa 的位 置上表達的是GC-2 cell 自己產生的 LRWD1 蛋白質，而本研究轉染 進GC-2 cell 的質體產生的預測大小為 100kDa 左右，因為分子量較大 較難表現，收取的目標蛋白質量很少，所以無法在圖上看到明顯片段。