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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53518

Title: 以 CRISPR/Cas9 基因編輯技術建立 dead end (dnd1) 標靶突變斑馬魚及淡水神仙魚之不孕控制技術
Targeted mutagenesis of dead end (dnd1) gene for infertility control of zebrafish and angelfish by CRISPR/Cas9 genome editing
Authors: Chu, Wai-Kwan
朱慧君
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 斑馬魚;神仙魚;原始生殖細胞;不孕技術;dead end;基因編輯;CRISPR/Cas9
zebrafish;angelfish;PGCs;infertility;genome editing;CRISPR/Cas9
Date: 2019
Issue Date: 2020-07-03T08:30:02Z
Abstract: dead end (dnd1) 編碼一種在脊椎動物上演化保守的 RNA 結合蛋白且專一 性表現在原始生殖細胞 (PGCs)。dnd1 在 PGCs 的遷移、存活及維持細胞命運扮 演至關重要的角色。本研究以 CRISPR/Cas9 基因編輯技術分別應用於斑馬魚及 神仙魚上建立 dnd1 基因標靶突變個體,探討建立不孕個體之可行性。斑馬魚作 為本研究中的模式動物,我們成功以 CRISPR/Cas9 基因編輯技術建立 dnd1 knockout 之斑馬魚並在 Drdnd1-/- 個體上達到完全不孕。dnd1 缺失斑馬魚胚胎 發育至 24 小時,已無法偵測到具生殖細胞專一性基因 vasa 之表現訊號。而在 性成熟個體上,Drdnd1+/- 無論雌雄與 wild type 斑馬魚在生殖腺組織、生殖細胞 專一性表現基因水平及交配模式並無顯著差異; 而在 Drdnd1-/- 個體上則發現全 雄性的現象,精巢結構與 wild type 斑馬魚相比明顯萎縮且缺失生殖細胞,但這 些個體仍能達到性成熟且具雄性性徵,能追尾讓雌性斑馬魚產卵,卻因無法產生 精子而未能成功使卵受精。在斑馬魚上我們成功從建立 dnd1 knockout 品系至檢 驗 knockout 個體是否不孕建立一套完整系統,期盼後續能應用於神仙魚上。而 在神仙魚的部分,本研究成功建立 Psdnd1 基因之 cDNA 全長,由於 dnd1 在 演化上極具保守性,我們透過與其他脊椎動物多重排比預測神仙魚 DND1 能性 區域。 Psdnd1 專一性表現在生殖腺組織且卵巢表現量較精巢表現高。同時我們 初步了解神仙魚胚胎發育過程並以描繪方式進行紀錄,首次突破神仙魚卵殼剝除 並建立神仙魚胚胎之原位雜交技術,揭示 Psdnd1 mRNA 在二細胞期便存在於分 裂溝。本研究成功以 CRISPR/Cas9 基因編輯技術應用於神仙魚,建立三種 Psdnd1 knockout 之 F0 世代,透過 F0 剪鰭分析篩選出優先配對分析的個體。 我們發現 Psdnd1 gRNA-1 較 Psdnd1 gRNA-2 在體細胞上有較強 knockout 結果; 可惜的是 Psdnd1 gRNA-1 & 2 兩個 gRNAs 共同作用下,大部分個體之定序結 果顯示缺失片段範圍超越預期,大片段掉落致失去 Exon-intron 之 splicing site, 導致無法預測胺基酸轉譯之結果,故我們傾向不使用兩個 gRNA 共同作用之組 別。雖然目前只有少部分 F0 個體達致性成熟並交配繁殖,透過 F1 剪鰭分析, 已鎖定某些能遺傳 knockout 序列且產生終止密碼子之 F0 個體。目前我們已成 功篩選出 3 隻 Psdnd1+/- 及 1 隻 Psdnd1-/- 個體,將於後續進行互配建立穩定 之 Psdnd1 標靶突變品系並進一步進行分析探討在神仙魚上達到不孕的可能性。
Dead end (dnd1) gene encoding an RNA binding protein with conserved RNA recognition motif (RRM) is specially expressed in primordial germ cells (PGCs) and germ cells of vertebrates. The DND1 is essential for PGCs migration, survival and may also play an important role in maintaining PGCs cell fate. In this study, we report targeted mutagenesis of dnd1 gene in the zebrafish model and ornamental freshwater angelfish (Pterophyllum scalare) by genome editing to obtain dnd1 knockout zebrafish and angelfish to explore the possibility of 100% infertility. We effectively performed targeted mutagenesis of zebrafish dnd1 gene in the F0 generation by genome editing technology resulted pre-mature stop codon due to insertion or deletion in exon 2 attacked by single gRNA. Heterozygotes and homozygotes of dnd1 knockout zebrafish are both selected by genotyping in F1 and F2 generation. Through whole-mount in situ hybridization, F2 zebrafish embroyos resulted loss of germ cell specific marker vasa signal in homozygotes. Based on the histological analyses of gonad between wildtype male and homozygotes dnd1 knock-out zebrafish, dnd1 knock-out resulted in loss of germ cells, but no difference between wildtype and heterozygotes zebrafish. The adult zebrafish with homozygotes dnd1 knockout are all male with courtship behavior to make wildtype female zebrafish spawning. The spawned eggs were found unfertilized then all died within 10 hours. We successfully set up a model to reveal whether dnd1 knockout individuals are infertile, and hope that the subsequent application can be applied on angelfish. In adult angelfish, Psdnd1 was only expressed in the gonad, with much higher expression in the ovary compared with the testis. We succeeded in establishing the embryogenesis stage and de-chorionation of angelfish embryos and first visualized the early PGCs expression patterns of Psdnd1 by whole mount in situ hybridization. We effectively preformed targeted mutagenesis of angelfish dnd1 gene in the F0 generation by CRISPR/Cas9 genome editing technology resulting in pre-mature stop codon due to insertion or deletion in exon 2 attacked by two single gRNA, respectively or two gRNAs in the same time via microinjection into one-cell fertilized eggs of angelfish. Heterozygotes and homozygotes of dnd1 knockout angelfish are both sucessfully selected by genotyping in F1 generation. We will confirm infertility in homozygotes later on. Targeted mutagenesis of critical PGCS genes such as dnd1 by genome editing can be applied in infertility control of transgenic fluorescent fish for GMO regulation and even non-transgenic precious ornamental fish breeds.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010633050.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53518
Appears in Collections:[水產養殖學系] 博碩士論文

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