Def-2, -3, -6 and -8, novel mouse genes differentially expressed in the haemopoietic system

To identify developmentally regulated genes during myeloid differentiation, a self-inactivating retroviral gene-trap vector carrying a beta-galactosidase-neomycin (SA/lacZ/neo) fusion gene was constructed and used to infect myeloid progenitor cells (FDCP-Mix A4). G418-resistant and beta-galactosidase positive cell lines (gene-trap integration [GTI] clones) were established and induced to differentiate in vitro into either macrophages or granulocytes. Expression of the trapped loci was monitored at a single-cell level by analysing the mature cell types for beta-galactosidase activity. All 37 GTI clones tested showed down-regulation either during granulocyte or both granulocytic and macrophage differentiation. The endogenous coding regions fused to the SA/lacZ/neo reporter gene were isolated from eight clones. Molecular analysis revealed that half of them represented novel mouse genes (def-2, -3, -6 and -8) which we confirmed to be differentially expressed in primary haemopoietic tissues. Database searches revealed no significant similarities for def-2 (associated with haemopoietic progenitors) and def-8 (expressed most strongly in peripheral leucocytes). Def-6, which is down-regulated upon the differentiation into myeloid as well as erythroid lineages, was found to be closely related but not identical with the recently described B-cell-specific switch recombinase SWAP-70. Def-3, which is down-regulated upon differentiation into granulocytes but expressed in progenitor cells and macrophages, defines a novel family of RNA binding proteins.     

Expression of Envelope Proteins of Endogeneous C-type Retrovirus on the Surface of Mouse and Human Oocytes at Fertilization

Retrovirus genomes express, among other products, the envelop (env) proteins SU (gp70) and TM (p15E). They coexist at the viral surface membrane and are able to promote immunosuppression and membrane fusion. In mouse oocytes, endogeneous retroviruses (ERV) genomes are expressed at fertilization, and antigen epitopes of the Moloney murine leukemia virus (MuLV) env protein gp70 are recognized in the cytoplasm of the oocytes before but not after fertilization. By using a panel of monoclonal antibodies (mAbs) raised against env components, we checked with laser scanning confocal microscopy (LSCM) whether gp70 and p15E were expressed also on the oocyte surface membrane (oolemma). Since we found that both mouse and human unfertilized oocytes expressed these ERV proteins on the oolemma and that the expression enfeebled significantly after fertilization, we assume that ERV genomes could play a ro^le at the sperm-egg binding and fusion.     

核心蛋白聚糖真核表达载体及逆转录载体的构建及鉴定

目的：通过获取核心蛋白聚糖（ｄｅｃｏｒｉｎ,ＤＣＮ）基因,构建ＤＣＮ真核表达载体和ＤＣＮ逆转病毒载体,以探索今后肾脏疾病基因治疗的途径。方法：从大鼠肾脏组织中抽取ＲＮＡ,经逆转录－ＰＣＲ方法,扩增核心蛋白聚糖ｃＤＮＡ,并经序列测定正确后,将ＤＣＮ插入真核表达载体ｐｃＤＮＡ３,应用Ｌｉｐｏｆｅｃｔａｍｉｎｅ介导将ｐｃＤＮＡ３－ＤＣＮ转染真核细胞ＣＯＳ－７细胞,并于转染后４８、７２和９６ｈ用ＥＬＩＳＡ     

异嗜性和多变性逆转录病毒受体 1在乳头状甲状腺癌患者中表达的意义

目的：探索异嗜性和多变性逆转录病毒受体1（ XPR1）基因在乳头状甲状腺癌（PTC）患者中表达的价值及其参与PTC发生发展的潜在通路。 方法：通过HPA数据库和UALCAN数据库探索XPR1在PTC组织及正常组织中的表达水平；通过cBioPortal数据库获取PTC患者临床资料及 XPR1基因表达值，探索 XPR1表达水平与患者性别、年龄、PTC分型、T分期、N分期、M分期和疾病分期的相关性；Cox回归分析探索PTC患者预后的影响因素；利用cBioPortal数据库探索 XPR1基因组学改变；基因本体（GO）和京都基因与基因组百科全书（KEGG）分析探索 XPR1参与PTC的生物学途径。 结果：HPA数据库分析发现，与正常组织相比，XPR1在PTC组织中表达增加；UALCAN数据库分析发现， XPR1基因表达量在PTC组织中较正常组织明显增多（ P<0.01），其中高柱亚型表达量最高，滤泡亚型表达量最低。PTC患者的 XPR1基因表达水平与患者年龄、PTC分型、T分期、N分期和疾病分期相关（ P<0.05或 P<0.01），而与性别和M分期不相关（均 P>0.05）。回归分析结果显示， XPR1表达水平可作为PTC患者预后的独立影响因子（ HR=2.894， P<0.05）。cBioPortal数据库显示，6%的PTC患者出现 XPR1基因突变，主要表现为错义突变，突变位点为蛋白结构域E615K。富集分析结果显示， XPR1可能通过参与调节代谢通路影响PTC发生发展。 结论： XPR1在PTC组织中表达增加，且影响患者预后，可能通过参与代谢通路影响PTC的发生发展，可作为PTC诊断和治疗的潜在靶点。     

一种高效逆转录病毒载体pSIV-1的构建及其功能的初步鉴定

构建高效的逆转录病毒载体pSIV-1,并对其功能进行初步鉴定。对pEGZ-Term、pLXSN、pGEX-5X-3等各类表达载体的调控元件进行分析后,运用分子生物学手段去除表达载体pEGZ-Term中的部分元件,定向插入启动子SV40、选择性标志G418、多克隆酶切位点等,从而获得一种新型逆转录病毒载体pSIV-1。运用新载体构建含人PD-L1基因片段的重组载体,并用脂质体转染法,将其转染至包装细胞系293T。收集含重组病毒的上清转染L929细胞,用含G418的培养液筛选,成活细胞经流式细胞仪检测到目的蛋白后,扩大培养,从而获得L929/PD-L1基因转染细胞。L929/PD-L1基因转染细胞与活化T细胞体外混合培养,观察PD-L1信号对T细胞活化后表达CD25、CD69的影响。结果,成功构建了pSIV-1逆转录病毒载体;酶切图谱分析与理论值一致;基因转染细胞能持续稳定高表达PD-L1,筛选时间较短,感染效率较高;PD-L1基因转染细胞能显著抑制T细胞的进一步活化。因此构建成一种新型高效逆转录病毒载体pSIV-1,为构建基因转染,并开展相关功能学研究奠定了良好的物质基础。