Factors affecting porcine sperm mediated gene transfer
Factors affecting porcine sperm mediated gene transfer
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DOI：
10.1016/j.rvsc.2010.09.015
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发表时间：
2011-12-01
影响因子：
2.4
通讯作者：
Gadea, Joaquin
中科院分区：
文献类型：
--
作者：
Alberto Garcia-Vazquez, Francisco;Ruiz, Salvador;Gadea, Joaquin
“精子介导的基因转移”（SMGT）是基于精子细胞结合外源DNA的能力。本研究的主要目的是通过SMGT提高转基因猪的产量。考虑到SMGT研究缺乏可重复性，外源DNA的结合和内化机制是一个尚未解决的问题，评价了SMGT法生产转基因动物所涉及的不同因素。在这里，我们着手：（1）评估DMSO处理后精子结合外源DNA的能力;（2）确定转基因-精子相互作用的位置;和（3）评估通过用DNA孵育的精子进行深度子宫内人工授精（AI）生产转基因仔猪的效率。在共培养2 h后，DNA结合率高于30%，但用DMSO（0.3%或3%）处理精子对DNA结合率无影响。精子质膜的完整性在DNA相互作用中起着关键作用，并且改变的质膜促进与外源DNA的相互作用。DNA主要与活力降低的精子结合。DNA分子主要分布在顶体后区（61.9%）。在宫内深Al后，共获得29头仔猪，但它们中没有一头整合了转基因。总之，虽然已经证实DNA可以与公猪精子结合，但本实验并未证实Al产生转基因猪的效率，这主要是由于DNA与功能精子的结合减少。(C)2010爱思唯尔有限公司保留所有权利。
"Sperm mediated gene transfer" (SMGT) is based on the ability of sperm cells to bind exogenous DNA. The main objective of this study was to improve the production of transgenic pigs by SMGT. Taking into account that there is a lack of repeatability in studies of SMGT and that the mechanism of binding and internalization of exogenous DNA is a question that has not been solved, different factors involved in the production of transgenic animals by SMGT method were evaluated. Here we set out to: (1) evaluate the sperm capacity to bind exogenous DNA after DMSO treatment; (2) determine the location of the transgene-spermatozoa interaction; and (3) evaluate the efficiency of production of transgenic piglets by deep intrauterine artificial insemination (AI) with sperm incubated with DNA. The percentage of DNA binding was higher than 30% after 2 h of co-culture, but it was not affected by sperm treatment with DMSO (0.3% or 3%). The integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions with exogenous DNA. DNA bound mainly to spermatozoa with reduced viability. DNA molecules were found to be mainly associated to the post-acrosomal region (61.9%). After deep intrauterine Al a total of 29 piglets were obtained, but none of them integrated the transgene. In conclusion, although it has been confirmed that DNA can associate with boar spermatozoa, the efficiency of producing transgenic pigs by Al was not confirmed by the present experiments, mainly due to a reduced DNA binding to functional spermatozoa. (C) 2010 Elsevier Ltd. All rights reserved.