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首页> 外文期刊>Journal of Neuroscience Methods >Quick sex determination of mouse fetuses.
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Quick sex determination of mouse fetuses.

机译：快速确定小鼠胎儿的性别。

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摘要

We designed a rapid, simple and accurate PCR method to determine sexual identity of mouse fetuses collected on embryonic day 15. A multiplex PCR amplification was used to detect male-specific sequence (Sry) in DNA extracted from fetal livers through SDS denaturation followed by high salt extraction and precipitation. This extraction method resulted in sufficiently purified DNA in < 1 h and was suitable for PCR. The DNA obtained was amplified using a robot thermal cycler for 33 cycles. The reaction was performed in 50 microl, using two sets of primers specific for Sry gene (chromosome Y) and IL3 gene (chromosome 11). Amplification duration was 1.5 h. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The 402 bp band (Sry) obtained identifies the male fetuses and the 544 bp product (IL3) confirms the correct amplification of the template DNA. The entire procedure took < 4 h. The specificity of the method was confirmed by fluorescent in situ hybridization using a specific male probe on cultured male and female neural stem cells. This method allowed the preparation and culture of pure male and female neural stem cells from fetal tissue.
机译：我们设计了一种快速，简单和准确的PCR方法，以确定在胚胎第15天收集的小鼠胎儿的性别同一性。采用多重PCR扩增检测通过SDS变性然后高SDS变性从胎儿肝脏中提取的DNA中的男性特异性序列（Sry）。盐提取和沉淀。此提取方法可在不到1小时的时间内得到足够纯化的DNA，适用于PCR。使用机器人热循环仪将获得的DNA扩增33个循环。使用两组对Sry基因（染色体Y）和IL3基因（染色体11）具有特异性的引物，在50微升中进行反应。扩增持续时间为1.5小时。通过在高压下运行的3％琼脂糖电泳进行结果评估。获得的402 bp条带（Sry）鉴定出雄性胎儿，而544 bp产物（IL3）证实了模板DNA的正确扩增。整个过程耗时少于4小时。通过使用特异性雄性探针在培养的雄性和雌性神经干细胞上进行荧光原位杂交，证实了该方法的特异性。该方法允许从胎儿组织制备和培养纯的雄性和雌性神经干细胞。

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