Shakweer W M E, Hafez Y M, El-Sayed A A, Awadalla I M, Mohamed M I
Animal Production Department, Agricultural and Biological Research Division, National Research Centre, 33 El Bohouth St. (Former El-Tahrir St.), Dokki, Giza, P.O. 12622, Egypt.
Animal Production Department, Faculty of Agriculture, Cairo University, Giza, Egypt.
J Genet Eng Biotechnol. 2017 Jun;15(1):13-21. doi: 10.1016/j.jgeb.2017.04.001. Epub 2017 Apr 22.
This study aims to produce transgenic ovine spermatozoa bearing Ossimi sheep growth hormone (Os_GH) cDNA using different methods. The complete coding sequence of Os_GH has been registered in GenBank accession no. KP221575. The sequence of Os_GH cDNA has been subcloned into pmkate2-N expression vectors to construct Os_GH-pmKate2-N vector. Five groups of sperm uptake were submitted. All groups were incubated at 37 °C for 1 h: Control (sperm cells were incubated without vector), Traditional incubation (sperm cells were incubated with vector), Heat shock (sperm cells were incubated with vector at 4 °C for 20 min and heated for 2 min at 42 °C), Heat shock + Dimethyl sulfoxide (DMSO) (sperm cells were incubated with vector and supplemented with 3% of DMSO and then submitted to heat shock regime) and DMSO (sperm cells were incubated with vector and supplemented with 3% DMSO). The sperm genomic DNA in groups was extracted. The Os_GH-pmKate2-N vector was introduced efficiently into the head of sperm cells in all treated groups. Adding DMSO either with or without heat shock increased the sperm uptake. The progressive motility was reduced ( < 0.05) by 29.9% in heat shock group compared to the control. Adding DMSO improved ( < 0.05) the total and progressive motilities by 8.2% and 19.8%, respectively in heat shock group compared to the heat shock group without DMSO. The results documented the ability of ovine spermatozoa to uptake the exogenous vector. Also, sperm incubation with 3% DMSO is the best method to introduce the exogenous vector into spermatozoa without notable adverse effects on sperm motilities.
本研究旨在采用不同方法制备携带奥西米羊生长激素（Os_GH）cDNA的转基因绵羊精子。Os_GH的完整编码序列已在GenBank中注册，登录号为KP221575。已将Os_GH cDNA序列亚克隆到pmkate2-N表达载体中，构建Os_GH-pmKate2-N载体。设置了五组精子摄取实验。所有组均在37℃孵育1小时：对照组（精子细胞在无载体的情况下孵育）、传统孵育组（精子细胞与载体一起孵育）、热休克组（精子细胞与载体在4℃孵育20分钟，然后在42℃加热2分钟）、热休克+二甲基亚砜（DMSO）组（精子细胞与载体一起孵育，并添加3%的DMSO，然后进行热休克处理）和DMSO组（精子细胞与载体一起孵育，并添加3%DMSO）。提取各组精子基因组DNA。在所有处理组中，Os_GH-pmKate2-N载体均有效地导入了精子细胞头部。无论有无热休克，添加DMSO均增加了精子摄取量。与对照组相比，热休克组的精子前向运动率降低了29.9%（P<0.05）。与无DMSO的热休克组相比，在热休克组中添加DMSO分别使总运动率和前向运动率提高了8.2%和19.8%（P<0.05）。结果证明了绵羊精子摄取外源载体的能力。此外，用3%DMSO孵育精子是将外源载体导入精子且对精子运动能力无明显不利影响的最佳方法。
