Heier Jonathon, Takle Kendra A, Hasley Andrew O, Pelegri Francisco
Laboratory of Genetics, University of Wisconsin - Madison, 425-G Henry Mall, Madison, Wisconsin.
Dev Dyn. 2015 Oct;244(10):1300-12. doi: 10.1002/dvdy.24315. Epub 2015 Aug 24.
Whole genome duplication is a useful genetic tool because it allows immediate and complete genetic homozygosity in gynogenetic offspring. A whole genome duplication method in zebrafish, Heat Shock, involves a heat pulse in the period 13-15 min postfertilization (mpf) to inhibit cytokinesis of the first mitotic cycle. However, Heat Shock produces a relatively low yield of gynogenotes.
A heat pulse at a later time point during the first cell cycle (22 mpf, HS2) results in a high (>80%) frequency of embryos exhibiting a precise one-cell division stall during the second cell cycle, inducing whole genome duplication. Coupled with haploid production, HS2 generates viable gynogenetic diploids with yields up to 4 times higher than those achieved through standard Heat Shock. The cell cycle delay also causes blastomere cleavage pattern variations, supporting a role for cytokinesis in spindle orientation during the following cell cycle.
Our studies provide a new tool for whole genome duplication, induced gynogenesis, and cleavage pattern alteration in zebrafish, based on a time period before the initiation of cell division that is sensitive to temperature-mediated interference with centrosome duplication. Targeting of this period may also facilitate genetic and developmental manipulations in other organisms.
全基因组复制是一种有用的遗传工具，因为它能使雌核发育后代立即实现完全的基因纯合。斑马鱼中的一种全基因组复制方法——热休克法，涉及在受精后13 - 15分钟（mpf）期间进行热脉冲处理，以抑制第一次有丝分裂周期的胞质分裂。然而，热休克法产生的雌核发育体产量相对较低。
在第一个细胞周期的较晚时间点（22 mpf，HS2）进行热脉冲处理，会导致高比例（>80%）的胚胎在第二个细胞周期出现精确的单细胞分裂停滞，从而诱导全基因组复制。结合单倍体生产，HS2能产生有活力的雌核发育二倍体，其产量比通过标准热休克法获得的产量高出4倍。细胞周期延迟还会导致卵裂球分裂模式的变化，这支持了胞质分裂在后续细胞周期纺锤体定向中的作用。
我们的研究基于细胞分裂开始前对温度介导的中心体复制干扰敏感的时间段，为斑马鱼的全基因组复制、诱导雌核发育和卵裂模式改变提供了一种新工具。针对这一时期也可能有助于其他生物体的遗传和发育操作。
