Chromosome counting in the mouse and human zygote using low-invasive super-resolution live-cell imaging
Chromosome counting in the mouse and human zygote using low-invasive super-resolution live-cell imaging
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使用低侵入超分辨率活细胞成像对小鼠和人类受精卵进行染色体计数
DOI：
10.1101/2021.03.14.435348
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Yamagata Kazuo
中科院分区：
文献类型：
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作者：
Hatano Yu;Mashiko Daisuke;Tokoro Mikiko;Yao Tatsuma;Hirao Ryota;Kitasaka Hiroya;Fukunaga Noritaka;Asada Yoshimasa;Yamagata Kazuo
在植入前胚胎中，染色体数目异常会导致发育失败和妊娠率降低。传统的染色体检测方法需要活检，可以降低相关遗传疾病的风险;然而，细胞数量的减少也会降低妊娠率。因此，我们尝试使用超分辨率活细胞成像作为一种新的染色体计数方法，不减少细胞数量或活力，在小鼠（Slc：ICR）胚胎中计数染色体。我们在可以获得幼崽的条件下，通过向胚胎注射组蛋白H2 B-mCherry mRNA，在第一次有丝分裂时对40条染色体进行了计数;然而，结果往往低估了染色体数量，并因胚胎和时间点而异。因此，我们开发了一种通过CRISPR/dCas介导的活细胞荧光原位杂交来计数染色体的方法，靶向着丝粒区域的序列，使我们能够更准确地计数小鼠和人类胚胎中的染色体。本文介绍的方法可广泛应用于辅助生殖技术，例如用于家畜/人类的技术，作为一种在transfer.Impact StatementLow-toxic超分辨率观察之前评估胚胎染色体完整性的技术，可在不收集细胞的情况下对植入前胚胎进行染色体计数。
In preimplantation embryos, an abnormal chromosome number causes developmental failure and a reduction in the pregnancy rate. Conventional chromosome testing methods requiring biopsy reduce the risk of associated genetic diseases; nevertheless, the reduction in cell number also reduces the pregnancy rate. Therefore, we attempted to count the chromosomes in mouse (Slc:ICR) embryos using super-resolution live-cell imaging as a new method of chromosome counting that does not reduce the cell number or viability. We counted the forty chromosomes at the first mitosis by injecting embryos with histone H2B-mCherry mRNA under conditions by which pups could be obtained; however, the results were often an underestimation of chromosome number and varied by embryo and time point. Therefore, we developed a method to count the chromosomes via CRISPR/dCas-mediated live-cell fluorescencein situhybridization targeting the sequence of the centromere region, enabling us to count the chromosomes more accurately in mouse and human embryos. The methodology presented here may be broadly applied to assisted reproductive technologies, such as those used in livestock animals/human, as a technique for assessing the chromosomal integrity of embryos prior to transfer.Impact StatementLow-toxic super-resolution observation enables chromosome counting in preimplantation embryos without cell collection.