The Breed & Biotechnology Key Laboratory of Sheep in Bingtuan, Xinjiang Academy of Agricultural and Reclamation Science, Shihezi 832000, PR China.
Anim Reprod Sci. 2013 Jun;139(1-4):18-24. doi: 10.1016/j.anireprosci.2013.04.009. Epub 2013 Apr 24.
The aim has been to set up a rapid and accurate microarray assay using sandwich mode for sex determination of bovine preimplantation embryos. Twelve sequence-specific oligonucleotide capture probes used to discriminate 12 samples were spotted onto the aldehyde-modified glass slides by Arrayer. The 2 recognition probes used to identify coding regions of the sex-determining region of the Y chromosome gene (SRY) and β-casein (CSN2) reference gene were coupled with biotin. The assay was optimized by using genomic DNA extracted from blood samples of known sex individuals. Polymerase chain reaction (PCR) was used to amplify the fragments in the HMG box region of SRY gene and CSN2 gene with sequence-specific primers. The sex of samples was identified by detecting both the SRY and CSN2 genes simultaneously in 2 reaction cells of microarrays, with the male having SRY and CSN2 signals and the female only CSN2. The sex of 20 bovine preimplantation embryos was determined by oligonucleotide microarray. The protocol was run with a blind test that showed a 100% (82/82) specificity and accuracy in sexing of leukocytes. The bovine embryos were transferred into 20 bovine recipients, with a pregnant rate of 40% (8/20). Three calves were born at term, and 5 fetuses were miscarried. Their sexes were fully in accordance with the embryonic sex predetermination predicted by oligonucleotide microarray. This suggests that the oligonucleotide microarray method of SRY gene analysis can be used in early sex prediction of bovine embryos in breeding programs.
本研究旨在建立一种快速、准确的基于夹心杂交模式的微阵列分析方法，用于鉴定牛早期胚胎的性别。通过 Arrayer 将 12 种用于区分 12 个样本的序列特异性寡核苷酸捕获探针点样到醛基修饰的载玻片上。用于识别 Y 染色体性别决定区基因（SRY）和β-酪蛋白（CSN2）参考基因编码区的 2 种识别探针与生物素偶联。通过使用来自已知性别的个体血液样本提取的基因组 DNA 对该检测方法进行优化。聚合酶链反应（PCR）使用序列特异性引物扩增 HMG 盒区域的 SRY 基因和 CSN2 基因片段。通过在微阵列的 2 个反应孔中同时检测 SRY 和 CSN2 基因，来确定样本的性别，其中雄性具有 SRY 和 CSN2 信号，而雌性仅具有 CSN2 信号。通过寡核苷酸微阵列鉴定了 20 个牛早期胚胎的性别。该方案进行了盲测，结果显示白细胞性别鉴定的特异性和准确性均为 100%（82/82）。将牛胚胎移植到 20 头牛受体中，妊娠率为 40%（8/20）。3 头小牛足月分娩，5 头胎儿流产。它们的性别与寡核苷酸微阵列预测的胚胎性别完全一致。这表明，寡核苷酸微阵列分析 SRY 基因的方法可用于牛胚胎繁殖计划中的早期性别预测。
