Making gynogenetic diploid zebrafish by early pressure.
Making gynogenetic diploid zebrafish by early pressure.
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DOI：
10.3791/1396
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发表时间：
2009-06-30
期刊：
影响因子：
--
通讯作者：
Moens, Cecilia
中科院分区：
文献类型：
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作者：
Walker, Charline;Walsh, Greg S;Moens, Cecilia
二倍体真核生物的杂合性常常使遗传学研究变得麻烦。直接从杂合雌性产生可存活的纯合子二倍体后代的方法允许在加速的正向遗传筛选中直接筛选F1突变的雌性有害突变。Streisinger等人描述了制造雌性（纯合子）二倍体斑马鱼的方法，方法是用紫外线灭活的精子激活斑马鱼卵，并使用利用微管的物理处理（热或压力）阻止第二次减数分裂或第一次合子细胞分裂。“早期压力”（EP）方法阻断受精后不久发生的减数分裂II。EP方法产生的存活胚胎的比例很高，这些胚胎可以发育成任何性别的可育成虫。该方法产生的胚胎除了在减数分裂期间通过重组与着丝粒分离的胚胎外，所有位点都是纯合的。在EP胚中，着丝粒位点的纯合突变率在50%之间，端粒位点的纯合突变率低于1%。这种方法是从斑马鱼书中逐字复制的。
Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al. described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book.