Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene
Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene
复制标题
DOI：
10.1016/j.theriogenology.2015.07.003
复制
发表时间：
2015-10-15
期刊：
影响因子：
2.8
通讯作者：
Psenicka, Martin
中科院分区：
文献类型：
--
作者：
Linhartova, Zuzana;Saito, Taiju;Psenicka, Martin
鲟鱼是一种古老的鱼类，以其鱼子酱而闻名。如今，它们中的大多数都处于极度濒危状态。鲟鱼(Acipenser Ruthenus)是欧亚大陆常见的鲟鱼物种，体型较小，繁殖周期是鲟鱼中最快的。这些物种可以作为代孕生产的宿主：应用对于恢复极度濒危和繁殖周期极长的巨型鲟鱼物种具有价值。只产生供体配子的一个先决条件是有一个不育的宿主。在鱼类中常用的绝育技术，如三倍体或杂交技术，并不能保证鲟鱼的不育。或者，可以通过使用一种临时的生殖细胞排斥特异性基因来实现绝育，这种基因被一种反义吗啉寡核苷酸(MO)击倒。MO的靶基因是死端基因(Drid)，这是一种脊椎动物特有的基因，编码一种RNA结合蛋白，对原始生殖细胞(PGC)的迁移和生存至关重要。为此，使用了俄罗斯鲟鱼(Agdnd)的DND同源物，其在起始密码子区域与分离的斯特雷特DND(Ardnd)片段具有相同的序列。逆转录聚合酶链式反应证实，ArdnD基因只在两性的性腺中有组织特异性表达。将去除PGCs的DND-MO和标记PGCs的异硫氰酸荧光素(FITC)-生物素-葡聚糖一起注射到1-4细胞期胚胎的营养区。在对照组中，仅注射FITC以验证注射方法和标记PGCs。在优化MO浓度和体积注射的基础上，采用250亩MO对鲟鱼胚胎进行灭菌。FITC标记对照组受精后21d体腔内未见原始生殖细胞，而FITC标记对照组仅在生殖脊内可见原始生殖细胞。此外，通过组织学和原位杂交观察了MO处理和未处理鱼的体腔，发现在不同时期(受精后60天、150天和210天)，生殖腺的形态中都没有生殖细胞。综上所述，这些结果报告了第一个已知的有效的鲟鱼绝育方法。(C)2015年提交人。由爱思唯尔公司出版。
Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production: application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (drid) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-mu M MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization. (C) 2015 The Authors. Published by Elsevier Inc.