Baars Destiny L, Takle Kendra A, Heier Jonathon, Pelegri Francisco
Laboratory of Genetics, University of Wisconsin.
Laboratory of Genetics, University of Wisconsin; Department of Neurobiology, University of Massachusetts Medical School.
J Vis Exp. 2016 Dec 16(118):54492. doi: 10.3791/54492.
Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.
倍性操作可实现有用的转变，如二倍体转变为四倍体，或单倍体转变为二倍体。在斑马鱼（Danio rerio）中，特别是纯合雌核发育二倍体的产生在遗传分析中很有用，因为它能从单个杂合母体直接产生纯合子。本文描述了一种基于第一次细胞周期热脉冲（热休克2，HS2）的倍性加倍改良方案。通过抑制中心粒复制，该方法会在第二个细胞周期导致精确的细胞分裂停滞。精确的单周期分裂停滞与未受影响的DNA复制相结合，导致全基因组加倍。与该方法相关的方案包括卵子和精子采集、精子的紫外线处理、体外受精以及热脉冲以引起单细胞周期分裂延迟和倍性加倍。该方案的改良版本可应用于诱导其他动物物种的倍性变化。
