建立青鳉囊胚移植技术开辟鱼类异体生殖的新途径

Surrogate production (SP) is an emerging novel reproductive technology, referring to the production of progeny from one organism called donor by another called surrogate host. The best example is the production of rainbow trout by salmon. SP has great potentials for rapid breeding and conservation biology in fish and shellfish and attracts increasing interest. Current SP approaches include germ cell transplantation into the blastula embryos and fry or into gonads, which has led to varying extents of success. Blastulas are easy and robust for cell transplantation; and foreign BECs and PGCs after blastula transplantation (BT) are capable of migrating into the gonad and gamete production in adulthood. The BT approach is particularly well-suited for fish as a large number of embryos are easily available in many of these organisms. Our previous results showed that medaka blastula cells and cultured primordial germ cells (PGCs) can give rise to germline chimeras upon being transplanted into blastula embryos. In addition, knock down of medaka dnd by morpholino (MO) injection resulted in complete loss of primordial germ cell (PGC), whereas over expression of dnd by RNA injection significantly increased the number of PGC. This proposal aims to develop medaka blastula transplantation (BT) as a novel SP approach in fish. We will make use dnd MO PGC-free blastula as host for germline replacement and donors are: (1) blastula embryo cells (BECs) with dnd overexpression to greatly boost PGCs; (2) primordial germ cells (PGCs) from developing embryos and (3) Spermatogonail stem cells (SSCs) from the testis. We will further investigate the molecular mechanisms of donor derived germ cells development in allogeneic recipients. In this study, we will establish SP by BT in medaka, this technology will open up new avenues of research in genetic breeding and restoration and reproduction of endangered species in close relatives.

异体生殖是一种新兴的繁殖技术，它是由受体产生供体的后代，如以鲑鱼产生虹鳟。异体生殖在动物特别是鱼贝类的快速繁育和保护生物学中有着广阔的应用前景，吸引了众多学者的关注。异体生殖的方法包括：生殖细胞移植到囊胚胚胎、小鱼或成鱼性腺。鱼类大量产卵，特别适合以囊胚为受体开展细胞移植研究。我们的研究发现，青鳉的囊胚细胞或培养的原始生殖细胞，移植到青鳉囊胚能形成生殖细胞嵌合体。此外，敲降青鳉dnd基因导致原始生殖细胞完全缺失，而过表达dnd则显著增加原始生殖细胞的数目。本项目旨在研究青囊胚移植，开辟鱼类异体生殖的新方法。具体方法是：以dnd敲降而缺失原始生殖细胞的囊胚为受体，以过表达dnd的囊胚细胞，原始生殖细胞和生殖干细胞为供体。优化囊胚移植参数和条件，在青鳉实现生殖细胞置换的异体生殖，并探讨生殖细胞在受体体细胞环境发育和分化的机制。本项目的实现，将为水产遗传育种和濒危种质资源的保护和恢复提供基础。

生殖细胞移植介导的异体生殖技术是一种细胞工程育种新技术，在繁殖困难、繁殖周期长或者濒危物种中具有广阔的应用前景。我们在青鳉中，以过表达dnd增加原始生殖细胞数目的囊胚细胞为供体，以敲降dnd而导致缺失原始生殖细胞而不育的囊胚胚胎为受体，得到了全是来自供体的后代，实现了囊胚细胞移植介导的借腹怀胎技术。我们接着开展了不同种间的生殖细胞移植研究，以日本青鳉精巢中分离的精原细胞为供体，移植到不育的西里波斯青鳉小鱼受体，移植的生殖细胞能迁移到受体的性腺。我们以模式鱼青鳉鱼为工具，将其建立的成熟技术应用到经济鱼类的遗传育种中。我们建立了长江刀鱼的精巢体细胞系，该细胞系表达体细胞标记基因，不表达生殖细胞标记基因，将该细胞系和青鳉精原干细胞混合培养，在体外能诱导后者分化形成精子；此外我们还建立了马口鱼的精原干细胞系，该细胞系仅表达生殖细胞相关基因和干细胞标记基因，不表达性腺体细胞标记基因。这2种经济鱼类细胞系的建立，为将来开展经济鱼类的生殖细胞移植和诱导分化研究奠定了基础。. 我们同时开展了鱼类生殖发育相关的miRNA和相关基因的表达和功能研究。我们获得了青鳉的性腺表达miRNAs库，并验证了雌雄性腺差异表达的miRNAs和其靶基因；我们发现青鳉的igf1和igf3基因在性腺生殖细胞和体细胞都有表达，其共定位表达能区分2种不同的卵巢膜细胞；我们证明了sox3和gcnf基因是精子细胞的拟染色小体组成部分；我们研究了性别决定基因sox3和foxl2的表达模式；我们发现过表达bucky ball基因影响原始生殖细胞的数目;敲降oct4能影响胚胎眼、脑中枢神经的发育和血管的发生。我们还研究了经济鱼如梅童鱼和长江刀鲚的生殖细胞标记基因，以便将来开展它们的生殖细胞培养和移植的研究。例如，梅童鱼的vasa基因的3’UTR能标记青鳉胚胎的原始生殖细胞，长江刀鱼的boule和dazl基因具有差异明显的表达模式，dazl基因在卵巢的巴尔氏小体有表达，而boule没有表达。我们还和其他老师合作研究了miRNA对斑马鱼性腺的再生研究。

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