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首页> 外文期刊>Experimental Animals >Application of Oocyte Cryopreservation Technology in TALEN-Mediated Mouse Genome Editing
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Application of Oocyte Cryopreservation Technology in TALEN-Mediated Mouse Genome Editing

机译：卵母细胞冷冻保存技术在Talen介导的小鼠基因组编辑中的应用

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Reproductive engineering techniques, such as in vitro fertilization (IVF) and cryopreservation of embryos or spermatozoa, are essential for preservation, reproduction, and transportation of genetically engineered mice. However, it has not yet been elucidated whether these techniques can be applied for the generation of genome-edited mice using engineered nucleases such as transcription activator-like effector nucleases (TALENs). Here, we demonstrate the usefulness of frozen oocytes fertilized in vitro using frozen sperm for TALEN-mediated genome editing in mice. We examined side-by-side comparisons concerning sperm (fresh vs. frozen), fertilization method (mating vs. IVF), and fertilized oocytes (fresh vs. frozen) for the source of oocytes used for TALEN injection; we found that fertilized oocytes created under all tested conditions were applicable for TALEN-mediated mutagenesis. In addition, we investigated whether the ages in weeks of parental female mice can affect the efficiency of gene modification, by comparing 5-week-old and 8–12-week-old mice as the source of oocytes used for TALEN injection. The genome editing efficiency of an endogenous gene was consistently 95–100% when either 5-week-old or 8–12-week-old mice were used with or without freezing the oocytes. Thus, our report describes the availability of freeze-thawed oocytes and oocytes from female mice at various weeks of age for TALEN-mediated genome editing, thus boosting the convenience of such innovative gene targeting strategies.
机译：生殖工程技术，例如体外施肥（IVF）和胚胎或精子的冷冻保存，对于遗传工程小鼠的保存，繁殖和运输至关重要。然而，尚未阐明使用工程化核酸酶如转录活化剂样效应核酸酶（Talens），尚未阐明这些技术可以应用于产生基因组编辑的小鼠。在这里，我们展示了使用冷冻精子在小鼠中使用冷冻精子进行体外施肥的冷冻卵母细胞的有用性。我们检查了关于精子（新鲜与冷冻），施肥法（交配vs. IVF）和受精卵（新鲜与冷冻）的卵母细胞来源的并排比较;我们发现在所有测试条件下产生的受精卵母细胞适用于Talen介导的诱变。此外，我们调查了父母女性小鼠周数的年龄是否会影响基因改性的效率，通过将5周龄和8-12周龄小鼠与用于TALEN注射的卵母细胞来源的小鼠进行比较。当使用5周龄或8-12周龄小鼠或不冷冻卵母细胞时，内源基因的基因组编辑效率始终如一95-100％。因此，我们的报告描述了在Talen介导的基因组编辑的各个周龄的雌性小鼠中冻融卵母细胞和卵母细胞的可用性，从而提高了这种创新基因靶向策略的便利性。

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