Laboratório de Embriologia Molecular e Transgênese, Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, RS, Brazil.
Theriogenology. 2011 Nov;76(8):1552-60. doi: 10.1016/j.theriogenology.2011.06.027. Epub 2011 Jul 31.
The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by fluorescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼ 153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8-10% using naked DNA or liposomes to 40-45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT.
1）纳米转染剂是否比其他常见的转染方法更适合 SMGT；2）NanoSMGT 是否能够将外源性 DNA 分子传递给牛胚胎；3）埃洛石纳米管（HCNs）是否可以作为转染试剂来提高转基因的传递效率。实验共使用了 4 种转染系统：裸 DNA（无转染剂）、脂质体转染、纳米聚合物和埃洛石纳米管。通过常规和实时 PCR 以及荧光显微镜检测精子对质粒的摄取及其向胚胎的转移情况，并检测精子活力、胚胎发育和 EGFP 表达情况。结果表明，转染程序会影响精子摄取的质粒数量（P < 0.05），纳米聚合物的转染效率最高（每个精子约 153 个质粒）。处理组对精子活力或活力没有影响。纳米聚合物和 HCNs 处理组向四细胞期胚胎传递的质粒数量明显高于脂质体和裸 DNA 组（P < 0.05）。使用裸 DNA 或脂质体作为转染剂时，携带转基因的胚胎数量从 8-10%增加到 40-45%，使用纳米聚合物或 HCNs 作为转染剂时（P < 0.05）。在导致胚胎的囊胚形成率方面，不同转染程序之间没有显著差异。然而，在任何处理中都没有鉴定出表达 EGFP 的胚胎。因此，纳米转染剂提高了牛胚胎中转基因的传递效率，而对胚胎发育没有不良影响。据我们所知，这是首次通过 NanoSMGT 生产携带转基因的牛胚胎。
