The effect of a novel cryoprotective agent, carboxylated ε-poly-L-lysine, on the developmental ability of re-vitrified mouse embryos at the pronuclear stage
The effect of a novel cryoprotective agent, carboxylated ε-poly-L-lysine, on the developmental ability of re-vitrified mouse embryos at the pronuclear stage
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DOI：
10.1016/j.cryobiol.2014.01.008
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发表时间：
2014-04-01
期刊：
影响因子：
2.7
通讯作者：
Kashiwazaki, Naomi
中科院分区：
文献类型：
--
作者：
Shibao, Yurie;Fujiwara, Katsuyoshi;Kashiwazaki, Naomi
转基因动物通常通过将外源DNA显微注射到原核（PN）阶段的胚胎中来产生。PN胚胎也可用于敲除动物，因为人工核酸酶如锌指核酸酶或转录激活因子样效应物核酸酶现在可用于修饰靶基因。如果胚胎可以用多轮玻璃化冷冻，则剩余的胚胎可以重复使用。在这项研究中，我们研究了重复玻璃化冷冻小鼠胚胎在PN阶段的发育能力，使用Cryotop。还研究了一种新的冷冻保护剂（CPA），羧化ε-聚-L-赖氨酸（COOH-PLL），是否可用于小鼠胚胎的玻璃化冷冻。PN胚胎以二甲基亚砜（DMSO）和乙二醇（EG）作为保护剂进行玻璃化冷冻。加热后，一些胚胎被重新玻璃化多达三次。再玻璃化不影响存活率和体外发育能力。PN胚胎也用COOH-PLL代替DMSO玻璃化冷冻多达三次。经COOH-PLL和EG玻璃化冷冻后的胚胎仍能保持较高的存活和发育能力。然而，用COOH-PLL和EG玻璃化冷冻三次的胚胎显示出显著高于用DMSO和EG玻璃化冷冻三次的胚胎的发育能力（61.2 ± 3.1%）（44.2 ± 2.7%），这与新鲜胚胎的发育能力（70.0 ± 3.6%）相当。总之，我们的结果表明，小鼠PN胚胎的再玻璃化冷冻对胚胎的体外和体内发育没有不利影响。此外，COOH-PLL可作为CPA用于小鼠PN胚胎的玻璃化冷冻。(C)2014爱思唯尔公司All rights reserved.
Transgenic animals are generally produced by microinjection of exogeneous DNA into embryos at the pronuclear (PN) stage. PN embryos also can be used for knockout animals because artificial nucleases such as zinc-finger nuclease or transcription activator-like effector nuclease are now available for modification of the targeted gene. If the embryos can be vitrified with multiple rounds, the remaining embryos without microinjection can be reused. In this study, we examined the developmental competence of repetitively vitrified mouse embryos at the PN stage using Cryotop. It was also examined whether a new cryoprotective agent (CPA), carboxylated epsilon-poly-L-lysine (COOH-PLL), is available for vitrification of mouse embryos. PN embryos were vitrified with dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as CPAs. After warming, some embryos were re-vitrified up to three times. The re-vitrification did not affect survival and in vitro developmental ability. PN embryos were also vitrified with COOH-PLL instead of DMSO up to three times. The embryos re-vitrified with COOH-PLL and EG also maintained high survival and developmental ability. However embryos vitrified with COOH-PLL and EG at three times significantly showed higher developmental ability (61.2 +/- 3.1%) than those vitrified with DMSO and EG at three times (44.2 +/- 2.7%) which was equivalent to that of fresh embryos (70.0 +/- 3.6%). Taken together, our results show that re-vitrification of mouse PN embryos did not have a detrimental effect on the in vitro and in vivo development of the embryos. In addition, COOH-PLL is available as a CPA for vitrification of mouse PN embryos. (C) 2014 Elsevier Inc. All rights reserved.