Preparation and Amplification of Colony of Goat Transgenic Fetal Fibroblast and Mammary Gland Epithelial Cell with Human Lactoferrin Gene

[Objective]The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C1 which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0%,50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture,neo gene was as screened gene,genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [Result] Compared with non-conditioned culture medium,100% conditioned culture medium could greatly increase survived rate of single colony cells (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%). Compared with control,con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%),confluence time of amplification culture was significantly decreased (20-30 d). The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [Conclusion] The study provides a reliable method for separating transgenic cell,inserting and diagnosing ideal vector,and can save expense and time for transgenic animal production.

Biological significance of RNA-seq and single-cell genomic research in woody plants

RNA-seq and single-cell genomic research emerge as an important research area in the recent years due to its ability to examine genetic information of any number of single cells in all living organisms.The knowledge gained from RNA-seq and single-cell genomic research will have a great impact in many aspects of plant biology.In this review,we summary and discuss the biological significance of RNA-seq and single-cell genomic research in plants including the single-cell DNA-sequencing,RNA-seq and single-cell RNA sequencing in woody plants,methods of RNA-seq and single-cell RNA-sequencing,single-cell RNA-sequencing for studying plant development,and single-cell RNA-sequencing for elucidating cell type composition.We will focus on RNA-seq and single-cell RNA sequencing in woody plants,understanding of plant development through single-cell RNAsequencing,and elucidation of cell type composition via single-cell RNA-sequencing.Information presented in this review will be helpful to increase our understanding of plant genomic research in a way with the power of plant single-cell RNA-sequencing analysis.

Progress in Single Cell Sequencing Technology

Cells are the basic unit of life structure and life activities.Because of the complex micro-environment of cells,the content of components that play a key role is relatively small,so single-cell analysis is extremely challenging.In recent years,single-cell sequencing technology has been developed and matured.Single-cell sequencing can reveal the composition and physiological diversity of cells,and the existing single-cell separation technology,single-cell whole genome amplification technology,single The principles and applications of cell whole transcriptome amplification technology and single cell transcriptome sequencing are summarized and summarized.

Novel Methods in the Study of the Breast Cancer Genome: Towards a Better Understanding of the Disease of Breast Cancer

Rapidly developing sequencing technologies and bioinformatic approacheshave provided us with an unprecedented instrument allowing for an unbiased and exhaustive characterization of the cancer genome in genetic, epigenetic and transcriptomic dimensions. This review introduces recent excitingfindings and new methodologies in genomic breast cancer research. With this development, cancer genome research will illuminate new delicate interactionsbetween molecular networks and thereby unravelthe underlying biological mechanisms for cancer initiation and progression. It also holds promise for providing a molecular clock for the estimation of the temporal processes of tumorigenesis. These methods in combination with single cell sequencing will make it possible to construct a family tree elucidating the evolutionary lineage relationships between cell populations at single-cell resolution. The anticipatedrapid progress in genomic breast cancer research should lead to anenhanced understanding of breast cancer biology andguide us towardsnovel ways to ultimatelyprevent and cure breast cancer.

The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation Genetic Diagnosis of Chromosomal Abnormalities

Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA （15 pg） and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification （MDA） system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing （CNV-Seq） was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient＇s cohort for uterine transplantation,

Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.

Human Nail Clippings as a Source of DNA for Genetic Studies

Blood samples have traditionally been used as the main source of DNA for genetic analysis. How-ever, this source can be difficult in terms of collection, transportation, and long-term storage. In this study, we investigated whether human nail clippings could be used as a source of DNA for SNP genotyping, null-allele detection, and whole-genome amplification. From extracted nail DNA, we achieved amplicons up to a length of ~400 bp and >96% concordance for SNP genotyping and 100% concordance for null-allele detection compared to DNA derived from matched blood sam-ples. For whole-genome amplification, OmniPlex performed better than Multiple Displacement Amplification with a success rate of 89.3% and 76.8% for SNP genotyping and null-allele detection, respectively. Concordance was ~98% for both methods. When combined with OmniPlex whole-genome amplification, human nail clippings could potentially be used as an alternative to whole blood as a less invasive and more convenient source of DNA for genotyping studies.

LOH-profiling by SNP-mapping in a case of multifocal head and neck cancer

AIM: To introduce an approach for the detection of putative genetic host factors that predispose patients to develop head and neck squamous cell carcinomas(HNSCC).METHODS: HNSCC most often result from the accumulation of somatic gene alterations found in tumor cells. A cancer-predisposing genetic background must be expected in individuals who develop multiple cancers, starting at an unexpectedly young age or with little carcinogen exposure. Genome-wide loss of heterozygosity(LOH) profiling by single nucleotide polymorphism microarray mapping was performed in a patient with a remarkable history of multifocal HNSCC.RESULTS: Regions of genomic deletions in germline DNA were identified on several chromosomes with a remarkable size between 1.6 Mb and 8.1 Mb(mega base-pair). No LOH was detected at the genomic location of the tumor suppressor gene P53.CONCLUSION: Specific patterns of germline DNA deletions may be responsible for susceptibility to HNSCC and should be further analyzed.

单细胞分选与测序技术在环境微生物领域的应用及前景

随着对环境微生物生态系统认知的深化,对其深入研究的需求持续增加,传统的培养组学和环境DNA测序等研究手段已经难以满足对微生物物种和功能多样性的全面了解。单细胞分选与测序技术为环境微生物研究开创了全新的视角,成为该领域的热点与前沿。单细胞技术将微生物研究的焦点从群落水平深入到个体细胞层面,通过精准分选并测序单个微生物细胞,深入挖掘微生物的种间和种内多样性以及功能特性。本综述介绍单细胞技术在环境微生物研究中的应用,详细论述单细胞分选和基因组扩增的关键过程,并探讨单细胞测序数据解析过程中的生物信息学基本流程,为单细胞分选与测序技术在环境微生物研究中的发展提供借鉴和启示。

基于不同方法的微量细胞全基因组遗传变异检测和比较分析

旨在用不同方法对微量细胞全基因组遗传变异进行检测和比较分析。本研究首先以3、5、7、10个猪耳缘成纤维细胞为试验材料,利用不同方法提取微量细胞基因组,进行全基因组测序(whole genome sequencing,WGS),比较不同细胞数及不同提取方法之间的遗传变异检测性能。随后利用牛囊胚的5个和7个滋养外胚层细胞获得的基因组进行全基因组测序和SNP芯片技术的比较分析,每组均进行3次重复。结果表明,针对不同细胞数,基于全基因组扩增技术(whole genomic amplification,WGA)的MDA(multiple displacement amplification)方法均比其他方法的DNA产物浓度、测序质量及SNP检出性能效果更优。使用REPLI-g^((R)) Single Cell Kit扩增7、10细胞数的DNA浓度显著高于3、5细胞数,但质量评估的各项性能基本无显著差异,且不同细胞数检测到的SNP位点数量相似。5、7细胞数的Illumina Bovine GGP芯片SNP位点call rate分别为74.09%和81.52%。全基因组测序相较于芯片可以获得更丰富的遗传变异信息,但两种检测手段共同检测到的SNP以及基因型相同的位点数占比较低。综上所述,本研究系统比较了不同细胞数下不同微量细胞基因组提取方法以及不同全基因组遗传变异检测技术的各项性能,结果显示利用REPLI-g^((R)) Single Cell Kit扩增7细胞进行二代测序可得到较为准确且稳定的结果,本研究建立了一套较为可靠的家畜胚胎微量细胞样品基因组提取和遗传变异检测方案,为将来实现准确的胚胎基因组选择和胚胎质量评估具有重要的意义和价值。

单细胞精度的表达数量性状位点研究进展

表达数量性状位点(expression quantitative trait loci,eQTL)表示控制基因表达量的遗传变异位点。eQTL分析是后全基因组关联研究时代鉴定疾病相关遗传位点功能的重要方法,且取得了诸多重要发现。传统的eQTL分析基于全基因组测序结合整体RNA测序技术,细胞间差异的基因表达水平会被掩盖,从而无法鉴定细胞类型或状态依赖的eQTL,因此也难以解析特定环境下疾病相关遗传变异位点。近年来,随着单细胞转录组测序技术的发展和应用普及,基于单细胞转录组测序的eQTL (single-cell RNA sequencing-based eQTL,sc-eQTL)研究技术逐渐成为热点,其优势在于可以充分利用单细胞测序的分辨率和颗粒度挖掘细胞类型、细胞状态以及细胞动态依赖的表达变异位点,显著提升解析基因表达关联的遗传变异位点的能力,对人们探究复杂器官的形成以及疾病的发生、发展、干预和治疗具有重要意义。本文主要从sc-eQTL研究的发展、设计方案、建模策略以及面临的挑战等诸多方面综述近年来的研究进展,以期为科研工作者挖掘致病位点,解析基因调控提供全新的视角。

多组学分析预测双硫死亡相关基因在肾透明细胞癌中的作用

目的肾透明细胞癌的肿瘤微环境与双硫死亡之间的关系尚不明确,本研究旨在探索双硫死亡相关基因在肾透明细胞癌中的作用。方法选择15个双硫死亡相关基因,并且使用癌症基因组图谱肾透明细胞癌数据库、单细胞RNA测序、单细胞空间RNA-seq、基因突变、预后影响、通路富集、药物敏感性和体外实验来表征它们的表达谱。结果肿瘤微环境分析显示C1和C2亚型之间存在不同的免疫浸润和突变模式,且C1患者预后较差。发现5个双硫死亡相关基因(SLC7A11,ACTN4,IQGAP1,ACTB和DSTN)是肾透明细胞癌的独立预后因素。其中,DSTN是最有效的肿瘤抑制基因,在肿瘤样本中显著下调,尤其在癌相关成纤维细胞中明显下调。单细胞药物敏感性分析显示,DSTN high的癌相关成纤维细胞表现出较强的药物敏感性。最后,本研究证实了DSTN在肾透明细胞癌组织及细胞系中的异常下调。结论DSTN可能是肾透明细胞癌潜在的诊断和治疗靶点。

单细胞多组学技术和应用

单细胞多组学技术是指结合多种不同的生物学技术,对单个细胞进行多方面的分析和研究,从而获得更全面、更准确的单细胞数据。该技术包括单细胞基因组学、单细胞转录组学、单细胞蛋白质组学、单细胞表观组学等。单细胞多组学技术的发展为我们提供了一种更加精确理解生物体内复杂的细胞类型和功能的方式,尤其是对于异质性细胞群体中少数的特殊细胞类型如干细胞或罕见癌细胞,具有非常重要的应用价值。通过融合不同技术获得的信息,单细胞多组学可以更准确地描述单个细胞在多个生命事件和过程中的状态与变化,为生命科学的研究提供更加全面和深入的视角。本文系统概述了目前代表性单细胞多组学技术的发展现状,总结了其在生物学研究中的重要应用和巨大潜力。

不同单细胞全基因组扩增方法的比较及MALBAC在辅助生殖中的应用

单细胞全基因组扩增(whole genome amplification,WGA)是指在单细胞水平对全基因组进行扩增的新技术,其原理是将分离的单个细胞的微量全基因组DNA进行扩增,获得高覆盖率的完整的基因组后进行高通量测序,用于揭示细胞异质性。目前,WGA方法主要包括引物延伸预扩增(primer extension preamplification PCR,PEP-PCR)、简并寡核苷酸引物PCR(degenerate oligonucleotide primed PCR,DOP-PCR)、多重置换扩增(multiple displacement amplification,MDA)、多次退火环状循环扩增(multiple annealing and looping-based amplification cycles,MALBAC)等。本文对不同的单细胞WGA方法的原理及应用情况分别进行了阐述,并对其扩增效率进行评价和比较,包括基因组覆盖度、均一性、重现性、SNV(single-nucleotide variants)和CNV(copy number variants)检测力等。综合对比不同单细胞WGA方法后发现,MALBAC的扩增均一性最高、等位基因脱扣率最低、重现性最好,且对于CNV和SNV的检测效果最好。本文还阐述了MALBAC技术在人类单精子减数重组、非整倍体分析以及人类卵细胞基因组研究中的应用。

植物叶片基因组DNA快速提取方法

为了满足分子生物学研究中大量植株基因需要PCR检测的需求,建立了一种无需研磨、无需有机试剂抽提的快速提取植物叶片基因组DNA的方法。通过比较基因组DNA的浓度及PCR检测结果,获得了最佳提取条件,即约50 mg叶片加入150μL含1%β-巯基乙醇的TE提取液,快速破碎1 min。破碎后的样品4℃13 500×g离心1 min,上清于-20℃冷冻后室温融解,4℃、13 500×g离心1 min,离心后收集的上清溶液即可用于PCR检测。整个提取过程仅需10-12 min,具有样品用量小、操作简单、廉价、高效等优点。使用此方法提取的烟草、水稻、大豆、玉米、油菜和花生的基因组DNA均可用于PCR扩增,并可成功扩增长度为3 244 bp的基因片段。此方法提取的基因组DNA也可用于对未知基因的扩增,获得4条新的花生actin基因序列。

低体积PCR扩增用于单细胞分离和检验

目的优化在单细胞分离检验中应用0.2mL试管做低体积扩增载体的最佳反应条件。方法制备理想口腔上皮细胞悬液,用0.2mL试管分别收集捕获到的5、10个细胞,设置蛋白酶K添加、PCR反应金牌酶用量、循环次数3组条件,用Identifiler誖Plus试剂盒进行复合扩增,比较各组的检出率、等位基因丢失率和非特异性扩增情况。结果用0.2mL试管做低体积扩增中,加蛋白酶K裂解、PCR反应金牌酶0.4μL、PCR反应32个循环,这3个条件的检出率较高,等位基因丢失率较低。结论在单细胞分离检验中采用0.2 mL试管进行低体积扩增,可以作为芯片-低体积扩增的有效补充手段。

一种实用的双链RNA病毒基因组克隆方法

以克隆水稻矮缩病毒(Rice dwarf virus.RDV)基因组片段 S11、S12为例.报道一种克隆植物dsRNA 病毒基因组的方法。具体过程为:利用 T4 RNA 连接酶将5′-磷酸、3′-氨基修饰的引物 Primer 1连接到 RDV 病毒基因组第11、12片段 dsRNA 的3′-OH 端,经逆转录、退火、补齐形成全长双链 cD-NA,使用单一的互补引物 Primer 2进行 PCR 扩增,扩增产物克隆在 pMD 18-T 载体上.对重组子进行两次限制性内切酶分析,结合序列测定分离鉴定 S11、S12。结果表明,这种方法能同时克隆 RDV 基因片段S11、S12,是一种有效实用的 dsRNA 病毒基因组克隆方法。

混合斑中精子细胞分离及其DNA制备方法

目的尝试建立一种检测混合斑中精子细胞的方法。方法使用显微操作法捕获精子细胞,全基因组扩增(多重置换扩增)精子细胞DNA。结果对10管精斑检材的全基因组扩增,获得了高产、保真的产物。使用50μL体系对20个精子细胞直接进行全基因组扩增,省去了对起始模板的纯化过程,DNA扩增倍数达30000倍以上,片段长度大多在15 kb以上,其STRs复合扩增分型结果有可参照性。结论显微操作法可以有效捕获精子细胞,排除干扰,多重置换扩增可以提供足够量的产物用于法医DNA分析,该方法具有可行性。

滚环DNA扩增技术及其应用

滚环扩增技术是一种等温信号扩增方法,DNA可在很短时间内实现指数扩增,因此,可用于痕量分子的检测。目前,该技术既可以扩增环状DNA、RNA,也可以扩增线性DNA,甚至全基因组DNA。目前,该技术主要用于全基因组扩增、核酸测序、单核苷酸多态性以及DNA芯片、蛋白质芯片分析等广泛领域。

激光显微切割分选少量胃癌细胞中PSCA基因的SNP分析

随着基因组关联分析方法的应用,越来越多与胃癌相关的易感基因被发现.易感基因的多态性检测已逐步进入胃癌临床诊断和研究.然而,利用少量胃粘膜细胞开展单核苷酸多态性(SNP)分析对胃癌进行早期诊断常遇下述困难,一是少量胃癌细胞混杂在多种细胞中,异常信号常易被淹没,二是细胞量极少,因此获得的基因组DNA量微,进行多位点或全基因组分析存在困难.本文利用激光显微切割技术分选少量胃癌细胞,结合全基因组放大技术,进行胃癌相关的前列腺干细胞抗原基因(PSCA)的SNP分析.通过聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和克隆测序方法分析,在分选的胃癌细胞中检测到PSCA的rs2976392位点胃癌相关的"A"等位与rs2294008位点胃癌相关的"T"等位.研究结果表明,所采用的全基因组放大方法保真性高,经过分选的胃癌细胞中SNP位点的检测灵敏度和可靠性大为提高.所建立的少量细胞基因多位点检测方法将同样应用于其它肿瘤和组织的少量细胞研究中,全基因组放大产物也可进行高通量的基因芯片和第二代测序研究.