在单细胞水平建立原位PCR基因鉴别技术平台——以精子基因型鉴别为例

Establishing Technology of in-situ PCR Genotype Identification in Single-celled Level

目的精子发生是一个包括有丝分裂和减数分裂的精细调控过程,这一过程是从精原干细胞开始在雄性睾丸的曲细精管中完成的,由哺乳动物雌雄性比例接近1∶1这一现象可以推知:受精时带X基因的精子和带Y基因的精子比率应该是1∶1,但事实上精子发生的过程中,分化出来的X、Y精子的比例是否也为1∶1并不清楚,因为精子发生过程中并不是所有细胞都能最终形成精子,然而要研究雌雄出生比率就需要先研究精子本身是否严格地按照1∶1形成,因此本实验通过建立快速可靠原位PCR技术平台为进一步研究精子发生的调控过程提供支持。步骤用HSL基因优化荧光原位PCR实验方案,包括蛋白酶K作用时间和浓度,原位PCR前先对精子进行解聚,再用SRY基因对精子进行原位PCR鉴定。结果在荧光显微镜下可清楚地看到精子头部的荧光信号,头部有绿色荧光信号的精子为带有SRY基因的Y染色体精子(Y精子),实验检测到昆明小鼠精子共493个,有信号的共273个,X∶Y=55.37%,经卡方检验,X精子与Y精子的实验结果趋近于1∶1。结论可利用荧光原位PCR技术在单细胞水平快速鉴定精子"性别",理论上可用于任何细胞的任何基因的鉴定。

Purpose Spermatogenesis is a fine control process with mitosis and meiosis,which is from spermatogonial stem cells began in male seminiferous tubule.By the phenomenon proportion of made to female in mammals,which is 1∶1,we can infer that the fertility rate of sperm with X gene and Y gene should be 1∶1.However,after differentiation,X and Y sperm for 1∶1 ratio is not known through spermatogenesis.Not all spermatocytes can develop into sperm during spermatogenesis eventually.However,to study the male and female birth ratio need to first study that if the spermatogenesis is strictly in accordance with 1∶1.Therefore,the present study,using a rapid and reliable platform of In-situ PCR,can further support the studies on regulation in spermatogenesis.Step Using HSL gene exploration steps,we can obtain an optimal fluorescence in-situ PCR experiment scheme,including proteinase K effect time and concentration,sperm depolymerization method and degree,etc,then applies to SRY gene.Result By Nikon fluorescence microscopy,we can clearly see the sperm head with fluorescent signals.Sperm head with green fluorescent signals target to SRY gene is the Y chromosome sperm（Y sperm）.Our lab tested 493 Kunming mice sperm altogether and there were total 273 signals.X sperm ∶ Y sperm = 55.37%.The experimental results approached to draw after the chi-square test.Conclusion We can use in-situ PCR technology to quickly appraisal the sperm ＂gender＂ in single-celled level,which is also suitable to detect any kind of gene in whatever the cell type is.