Application of Genome Editing Technology in Targeted Mutagenesis of Dnd1 Gene for the Infertility Control of Ornamental Fish( I )

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Project title

Code/計畫編號

MOST109-2622-B019-002-CC1

Translated Name/計畫中文名

以基因體編輯技術標靶突變dnd1基因在觀賞魚不孕控制之應用(1/3)

Project Coordinator/計畫主持人

Hong-Yi Gong

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Aquaculture

Website

https://www.grb.gov.tw/search/planDetail?id=13453943

Year

2020

Start date/計畫起

01-06-2020

Expected Completion/計畫迄

31-05-2021

Bugetid/研究經費

1800千元

ResearchField/研究領域

漁業

在全球水產和觀賞魚產業中，基因轉殖物種開發具有龐大的潛在商機。隨著消費者對觀賞魚飼養需求愈趨美觀且精緻化，新品種的開發能讓產業提升在觀賞魚市場上的競爭力。隨著基因轉殖技術的成熟，漸漸發展出許多新穎的「螢光魚」品系。但礙於基因轉殖物種因未能符合GMO法規要求而無法銷售。同時，若這些優化的物種一旦逃離養殖環境，流入自然環境自行繁殖或與野生種群雜交，都會對生態系統和環境構成極大威脅。因此，我們認為開發實用且有效的不孕技術，將能保護自然生態並降低基因轉殖物種所帶來的威脅，樹立環境有善且負責之養殖產業形象。為了探討不孕技術之建立，我們必須了解生殖細胞的發育機制。dead end1 (dnd1) 編碼一種在脊椎動物上演化保守及具RRM結構的 RNA 結合蛋白，且專一性表現在原始生殖細胞 (PGCs)和生殖細胞。而DND1對於PGCs的遷移、存活及維持細胞命運扮演至關重要的角色。根據過去的研究，我們利用單個gRNA針對Exon2進行標靶突變，因序列缺失或插入而導致提前產生終止密碼子，成功以 CRISPR/Cas9 基因編輯技術建立 dnd1 knockout 之斑馬魚。在F1和F2子代中，透過基因型分析成功篩選出Drdnd1+/- 及Drdnd1-/-個體。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透過 dnd1 knockout 達致不孕的效果，並為檢驗 knockout 個體是否不孕建立一套完整系統。本計劃目標為探討在神仙魚上，能否透過CRISPR/Cas9基因編輯技術針對dnd1基因進行標靶突變而導致不孕。因芝林企業在神仙魚繁養殖方面擁有豐富的經驗，能隨時予以協助和配合輔助計劃進行。此外，藉由本計劃，我們希望能將已開發之斑馬魚不孕技術應用至螢光斑馬魚上，以證此不孕技術之實用性。而與芝林企業配合的優勢，主要為更能讓我們真切了解產業應用之需求，從而使我們能進一步調整及優化不孕技術。 Transgenic species have great potential for both aquatic and ornamental fish industry globally. As the consumer’s demand for ornamental fish becomes more refined, unique and innovative, new species development may increase the industry’s competitiveness. With the maturity of transgenic technology, more and more new fluorescent species have been established. However, it is currently not possible to sell due to the source control and genetic modification regulations (GMO). Also, once these fish escape and interbreeding with wild stock, it poses a great threat to our ecosystem and environment. We believe that developing practical infertility control technology is the most effective way to avoid the ecological risks, also show support to the development of environmentally-responsible aquaculture. To establish an infertile individual, we must first understand the development mechanisms of germ cells. Dead end1 (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 our previous study, we successfully performed targeted mutagenesis of zebrafish dnd1 gene 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. Based on the histological analyses of gonad between wild-type male and homozygotes dnd1 knockout zebrafish, dnd1 knockout resulted in loss of germ cells, but no difference between wild-type and heterozygotes zebrafish. The adult zebrafish with homozygotes dnd1 knockout are all male with courtship behavior to make wild-type female zebrafish spawning. The spawned eggs were found unfertilized then all died within 10 hours. We successfully established infertile zebrafish by dnd1 knockout with CRISPR/Cas9, also set up a model to reveal whether dnd1 knockout individuals are infertile. In this proposal, we attempt to identify whether performing dnd1 knockout by CRISPR/Cas9 can cause infertile in angelfish. As JY LIN Group has extensive experience of farming and breeding angelfish, they may provide the advancement or support to optimize the scheme. In addition, we hope to establish fluorescent zebrafish with dnd1 knockout to prove the applicability of infertility control by genome editing. Base on the corporation with JY LIN Group, it may allow us to have the opportunity to improve the infertility control technology, as we are able to understand the needs of industry.

Keyword(s)

斑馬魚
神仙魚
原始生殖細胞
不孕控制
基因體編輯
CRISPR/Cas9
dnd1
zebrafish
angelfish
PGC
infertility control
genome editing

DSpace CRIS

Application of Genome Editing Technology in Targeted Mutagenesis of Dnd1 Gene for the Infertility Control of Ornamental Fish( I )

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