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基于单细胞转录组测序技术筛选牛体内囊胚性别特异性基因
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作者 崔宝山 黄飞 +2 位作者 王杰 李楠 高庆华 《中国畜牧兽医》 CAS 北大核心 2022年第12期1473-1481,共9页
【目的】探究牛体内雌性和雄性囊胚在mRNA水平上的特异性差异,挖掘雌性和雄性囊胚发育差异相关候选基因。【方法】参考GenBank中牛牙釉质基因(AMEL)序列(AMELX基因,登录号:NM_001014984.1;AMELY基因,登录号:NM_174240.2)设计引物,以胚... 【目的】探究牛体内雌性和雄性囊胚在mRNA水平上的特异性差异,挖掘雌性和雄性囊胚发育差异相关候选基因。【方法】参考GenBank中牛牙釉质基因(AMEL)序列(AMELX基因,登录号:NM_001014984.1;AMELY基因,登录号:NM_174240.2)设计引物,以胚胎内细胞DNA为模板,采用巢式PCR扩增技术鉴定单个牛体内囊胚的性别。选用确定性别的单个雌性和雄性囊胚为试验材料,采用Smart-Seq2扩增技术构建单个胚胎测序文库,应用Illumina HiSeq Xten高通量测序平台对单个胚胎进行单细胞转录组测序(scRNA-Seq),并进行了基因差异表达、GO功能和KEGG通路分析。【结果】通过巢式PCR扩增胚胎内细胞中的AMEL基因,确定了胚胎性别;基因表达分析筛选出两组之间差异表达基因(DEGs)6 160个,其中雌性特异性基因675个,雄性特异性基因305个;GO功能注释发现雌性特异性基因显著注释在核苷酸结合、信号转导调控、多细胞生物发育、细胞骨架等条目,雄性特异性基因显著注释在氧化磷酸化、线粒体、线粒体内膜、核糖体等条目;KEGG通路富集分析发现7条与雌性和雄性囊胚发育差异相关的通路,分别为代谢途径、糖酵解、磷酸戊糖途径、细胞衰老、氧化磷酸化、调节干细胞多能性的信号通路和Wnt信号通路;并利用GO和KEGG结果筛选出5个在牛体内囊胚期胚胎发育过程中具有直接或间接作用的基因:FBP1、GADD45G、FHL2、FOSB和WNT2B。【结论】牛体内雌性和雄性囊胚之间存在广泛的转录差异,性别特异性基因FBP1、GADD45G、FHL2、FOSB和WNT2B可能是直接或间接影响胚胎发育的关键基因。 展开更多
关键词 牛体内囊胚 单细胞转录组测序(scRNA-Seq) 性别特异性基因 富集分析
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Integrative analysis of in vivo recording with single-cell RNA-seq data reveals molecular properties of light-sensitive neurons in mouse V1
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作者 Jianwei Liu Mengdi Wang +7 位作者 Le Sun Na Clara Pan Changjiang Zhang Junjing Zhang Zhentao Zuo Sheng He Qian Wu Xiaoqun Wang 《Protein & Cell》 SCIE CAS CSCD 2020年第6期417-432,共16页
Vision formation is classically based on projections from retinal ganglion cells(RGC)to the lateral geniculate nucleus(LGN)and the primary visual cortex(V1).Neurons in the mouse V1 are tuned to light stimuli.Although ... Vision formation is classically based on projections from retinal ganglion cells(RGC)to the lateral geniculate nucleus(LGN)and the primary visual cortex(V1).Neurons in the mouse V1 are tuned to light stimuli.Although the cellular information of the retina and the LGN has been widely studied,the transcriptome profiles of single light-stimulated neuron in V1 remain unknown.In our study,in vivo calcium imaging and whole-cell electrophysiological patch-clamp recording were utilized to identify 53 individual cells from layer 2/3 of V1 as lightsensitive(LS)or non-light-sensitive(NS)by single-cell light-evoked calcium evaluation and action potential spiking.The contents of each cell after functional tests were aspirated in vivo through a patch-clamp pipette for mRNA sequencing.Moreover,the three-dimensional(3-D)morphological characterizations of the neurons were reconstructed in a live mouse after the whole-cell recordings.Our sequencing results indicated that V1 neurons with a high expression of genes related to transmission regulation,such as Rtn4r and Rgs7,and genes involved in membrane transport,such as Na+/K+ATPase and NMDA-type glutamatergic receptors,preferentially responded to light stimulation.Furthermore,an antagonist that blocks Rtn4r signals could inactivate the neuronal responses to light stimulation in live mice.In conclusion,our findings of the vivo-seq analysis indicate the key role of the strength of synaptic transmission possesses neurons in V1 of light sensory. 展开更多
关键词 light sensitivity vivo-seq patch-seq calcium imaging in vivo whole cell recording in vivo
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