SummaryThis study aimed to investigate the ability of disulphide-less crotamine (dLCr) to complex DNA and to evaluate whether the DNA–dLCr complex is capable of improving transfection in bovine embryos. Three experiments were performed to: (i) evaluate the formation and stability of the DNA–dLCr complex; (ii) assess the dLCr embryotoxicity by exposure of bovine embryos to dLCr; and (iii) assess the efficiency of bovine embryo transfection after microinjection of the DNA–dLCr complex or green fluorescent protein (GFP) plasmid alone (control). DNA complexation by dLCr after 30 min of incubation at 1:100 and 1:50 proportions presented higher efficiency (P < 0.05) than the two controls: native crotamine (NCr) 1:10 and lipofectamine. There was no difference between DNA–dLCr 1:25 and the controls. The DNA–dLCr complexation was evaluated at different proportions and times. In all, at least half of maximum complexation was achieved within the initial 30 min. No embryotoxicity of dLCr was verified after exposure of in vitro fertilized embryos to different concentrations of the peptide. The effectiveness of dLCr to improve exogenous gene expression was evaluated by microinjection of the DNA–dLCr complex into in vitro fertilized zygotes, followed by verification of both embryo development and GFP expression. From embryos microinjected with DNA only, 4.6% and 2.8% expressed the GFP transgene at day 5 and day 7, respectively. The DNA–dLCr complex did not increase the number of GFP-positive embryos. In conclusion, dLCr forms a complex with DNA and its application in in vitro culture is possible. However, the dLCr peptide sequence should be redesigned to improve GFP expression.

中文翻译：

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无二硫化物的克罗胺对 DNA-肽复合物的形成有效，但不能改善牛胚胎转染

总结本研究旨在研究无二硫化物的克罗胺 (dLCr) 复合 DNA 的能力，并评估 DNA-dLCr 复合物是否能够改善牛胚胎的转染。进行了三个实验： (i) 评估 DNA-dLCr 复合物的形成和稳定性；(ii) 通过将牛胚胎暴露于 dLCr 来评估 dLCr 胚胎毒性；(iii) 评估单独显微注射 DNA-dLCr 复合物或绿色荧光蛋白 (GFP) 质粒后牛胚胎转染的效率（对照）。dLCr 以 1:100 和 1:50 的比例孵育 30 分钟后的 DNA 复合效率更高（磷< 0.05) 比两个对照：天然 crotamine (NCr) 1:10 和 lipofectamine。DNA-dLCr 1:25 与对照之间没有差异。以不同的比例和时间评估 DNA-dLCr 络合。总之，在最初的 30 分钟内至少达到了最大络合度的一半。dLCr 暴露后未验证胚胎毒性体外受精胚胎到不同浓度的肽。dLCr 改善外源基因表达的有效性通过将 DNA-dLCr 复合物显微注射到体外受精卵，然后验证胚胎发育和 GFP 表达。从仅显微注射 DNA 的胚胎中，4.6% 和 2.8% 分别在第 5 天和第 7 天表达了 GFP 转基因。DNA-dLCr 复合物没有增加 GFP 阳性胚胎的数量。综上所述，dLCr 与 DNA 形成复合物及其在体外文化是可能的。然而，应重新设计 dLCr 肽序列以改善 GFP 表达。