建立以始基生殖細胞為媒介進行鳥禽保種之研究

Establishment of avian biodiversity preservation platform via primordial germ cells (PGCs)

陳奕臣(Yi-Chen Chen)

指導教授 ： 吳信志

共同指導教授 ： 潘博通(Bertrand Pain)

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2018年)

https://doi.org/10.6342/NTU201804355

摘要

始基生殖細胞為配子之前驅物，此等細胞可被視為種原保存之關鍵材料並被用於鳥類之遺傳多樣性管理。在始基生殖細胞移植後，為獲得配子及其子代，使用不具內源性生殖細胞之移植受體乃是理想之方式以提升被移植之始基生殖細胞之性腺傳承力。土番鴨乃公番鴨及母家鴨屬間雜交所得，此等雜交鴨雖具正常之體系發育，但配子發生時之缺陷乃致不育。因此，本試驗擬移植外源始基生殖細胞於土番鴨以探究其不育之成因及此鴨種成為生殖細胞移植通用受體之可能。
本研究之首要目的為分離並體外培養雞之始基生殖細胞，接著將此培養系統套用於培養不同鴨種之始基生殖細胞。首先，番鴨、北京鴨及雜交所得之土番鴨皆可被成功地分離出始基生殖細胞，細胞在體外增殖及遺傳修飾後其生殖系特性及發育潛力仍能維持。再者，始基生殖細胞被用以移植於土番鴨以進行配子發生之試驗。結果呈現，被始基生殖細胞移植之土番鴨無法產生卵或精子，但其精液中具有大量之細胞及細胞碎屑。此外，應用定量反轉錄聚合酶鏈鎖反應分析發現性腺體系中用來生成或降解維甲酸之酵素，其mRNA 表現量在發育過程之表現波動在親源鴨種及土番鴨中呈現非同步性。此等結果說明土番鴨性腺中之生殖細胞分化不全可能源於性腺發育時體細胞與生殖細胞間之交互作用。
本研究之次要目的為發展一套大量生產鳥禽類生殖細胞之方法。為此，試驗中建立了一種化學成分明確之培養液，可用以長期培養雞始基生殖細胞。此培養液結合功能性聚合物，發展為一種無需機械攪拌之三維懸浮培養系統以達到細胞量化生產之需求。量化生產之細胞易於冷凍保存，因此該系統可藉由擴增保存兩性別始基生殖細胞之遺傳資源進而貢獻於鳥類種原保存。此外，鳥禽類所展現之特殊醣基化修飾具醫療益處，成分明確之三維懸浮培養系統可做為雞始基生殖細胞之量化生產以達到生產重組蛋白及疫苗生產之目的。

關鍵字

種原保存 ； 雞 ； 鴨 ； 始基生殖細胞 ； 三維培養 ； 雜交不育

並列摘要

Primordial germ cells (PGCs) are the progenitors of gametes and could be seen as the key elements for germplasm preservation and the management of avian genetic biodiversity. To obtain the gametes and offspring, a recipient with no endogenous germ cells would be ideal to improve the germline transmission of the donor transplanted PGCs. In duck species, such bird exists as represented by the mule duck, produced by the intergeneric crossing between Muscovy drake (Cairina moschata) and female domestic duck (Anas platyrhynchos). This hybrid has normal somatic development but is impaired in the gamete production leading to hybrid sterility. Therefore, we aim to investigate the cause of this sterility and ultimately to test such breed as the universal recipient for transfer of exogenous duck PGCs to produce gametes of the donor genotype.
The first aim of the present project was to isolate and establish an in vitro culture of duck PGCs derived and adapted from the well-known chicken culture system. First, duck PGCs were successfully isolated from Muscovy, Pekin and hybrid mule ducks. Their germline characteristics and developmental potential were analyzed and were shown to be retained after being expanded in vitro and genetically modified. Second, those PGCs were transplanted to test the gametogenesis in mule duck but no sperm or ovum was produced in transplanted mule ducks. Instead, a large number of cells and debris was found in the semen collected from the mule drakes with PGC transplantation. Among others, mRNA levels of the gonadal somatic enzymes for retinoic acid (RA) synthesis and degradation showed asynchronous dynamic patterns in different developmental stages among parental species and mule ducks. Those observations could lead to the hypothesis of an impairment of the germ cell differentiation in the mule duck gonad that could be due to an altered germ-somatic cell-cell interaction leading to the sterility of the transplanted mule duck.
The second aim of the project was to develop a process of large-scale amplification of avian germ cells. For that, a chemically defined medium was established to grow and amplify chicken PGCs in long-term culture. By adding a functional polymer in the medium, a three-dimensional (3D) suspension culture without mechanical stirring was established leading to a scalable production of PGCs. This system could also contribute to germplasm preservation by amplifying the genetic resource as PGCs of avian species in both genders. Those large-scale produced cells could thus be easily cryopreserved. Additionally, avian specific glycosylation has attracted therapeutic interests and the defined 3D suspension culture system could provide a useful platform for large-scale production of avian PGCs to produce recombinant proteins and vaccines.

並列關鍵字

germplasm preservation ； chicken ； duck ； primordial germ cell (PGC) ； three-dimensional (3D) culture ； hybrid sterility

參考文獻

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