Genomic targeting by recombinase‐mediated cassette exchange in the spotted wing drosophila,Drosophila suzukii

Drosophila suzukii is a significant pest of stone and small fruits. The genome of this species has been sequenced and manipulated by transposon‐mediated transformation and CRISPR/Cas9 gene editing. These technologies open a variety of possibilities for functional genomics and genetic modifications that might improve biologically based population control strategies. Both of these approaches, however, would benefit from genome targeting that would avoid position effects and insertional mutations associated with random transposon vector insertions, and the limited DNA fragment insertion size allowed by gene editing. Here, we describe an efficient recombinase‐mediated cassette exchange (RMCE) system for D. suzukii in which heterospecific lox recombination sites were integrated into the genome by transposon‐mediated transformation and subsequently targeted for double recombination by a donor vector in the presence of Cre recombinase. Three loxN/lox2272 landing site lines have previously been created in D. suzukii, and quantitative PCR determined that polyubiquitin‐regulated enhanced green fluorescent protein expression is least susceptible to position effect suppression in the 443_M26m1 line. We presume that RMCE target sites may also be inserted more specifically into the genome by homology‐directed repair gene editing, thereby avoiding position effects and mutations, while eliminating restrictions on the size of donor constructs for subsequent insertion.     

Expression of exogenetic enhanced green fluorescent protein in rat endocranium through lentivirus infection

The study aims to investigate whether exogenetic green fluorescent protein is able to express in the endocranium of rats, and to establish a method for further study in exogenetic gene knock-in or gene overexpression. Forty female Sprague Dawley (SD) rats were randomly divided into 4 groups with 10 in each: low and high dose groups, treated with 10% and 100% EGFP-lentivirus, respectively; negative control group, treated with virus enhancer; sham group, treated with normal saline. Seven days later, half rats’ brain tissues were perfusion fixed and fresh brain tissues were obtained from the rest after euthanasia in each group. Immunohistochemical analysis, Western blotting and RT-PCR were respectively performed to detect the site where EGFP expressed and its levels. Immunohistochemical analysis demonstrated that EGFP was successfully expressed in brain tissue of those rats infected with EGFP-lentivirus. Both Western blotting and RT-PCR showed that EGFP was expressed after treatment with EGFP-lentivirus, and the expression level increased with the dosage of the vector. Exogenetic EGFP gene can express in brain tissue of the rat, which laid a solid foundation for future studies in exogenetic gene knock in or gene overexpression.     

Protein tyrosine phosphatase conjugated with a novel transdermal delivery peptide,astrotactin 1–derived peptide recombinant protein tyrosine phosphatase (AP-rPTP), alleviates both atopic dermatitis–like and psoriasis-like dermatitis

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EGFP和CM-Dil示踪骨髓间充质干细胞构建组织工程骨的体内研究

目的:应用增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)和CM-Dil标记技术,观察组织工程骨在体内形成过程中种子细胞的变化和转归.方法:分别用EGFP慢病毒表达和CM-Dil染料的方法标记比格犬骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs),MTT法检测标记细胞的体外增殖能力.BMSCs接种珊瑚支架体外成骨诱导7天后,将未标记组、EGFP组和CM-Dil组分别植入裸鼠背部皮下,空白支架作为阴性对照.术后4、8、12周取材,HE染色观察成骨情况,EGFP组采用GFP免疫组化、CM-Dil组冰冻切片荧光显微镜下示踪BMSCs在体内的变化.结果:两种标记技术能高效标记BMSCs,标记前后细胞的体外增殖无显著性差异(P>0.05).细胞-支架复合物植入体内12周后有新生骨形成,标记细胞数量随时间延长而逐渐减少,12周后仍显示有部分标记细胞存活.结论:EGFP和CM-Dil可用于示踪组织工程种子细胞,通过示踪说明BMSCs在体内组织工程骨成骨过程中发挥了重要作用.     

凋亡素基因对人结肠癌细胞作用的研究

目的探讨凋亡素基因的导人对人结肠癌细胞的影响。方法将凋亡素基因VP3克隆人真核表达载体pEGFP-C2，构建成重组质粒pEGFP-VP3。用脂质体转染法将pEGFP-C2和pEGFP—VP3分组分别转染人类结肠癌细胞（Lovo）和小鼠胚胎成纤维细胞（NIH3T3），通过荧光显微镜观察绿色荧光蛋白在不同组细胞中的定位、表达及细胞的生长、凋亡情况，MTT法测定不同组细胞的生长曲线，并用AnnexinV—FITC流式细胞技术检测细胞的凋亡率。结果在Lovo／EGFP组和3T3／EGFP—VP3组细胞中，EGFP均匀分布于细胞中，细胞形态无明显变化，细胞凋亡率较非转染细胞组亦无明显增加。而在Lovo／EGFP-VP3组细胞中，EGFP-VP3以荧光颗粒形式集中在细胞核，并逐渐变粗，最后细胞碎裂成片状，流式细胞技术检测Lov0／EGFP-VP3组的细胞凋亡率明显高于其他对照组（P〈0．01）。结论pEGFP-VP3可诱导人类结肠癌细胞凋亡。     

Enhanced self-renewal capability in hepatic stem/progenitor cells drives cancer initiation

BACKGROUND & AIMS: Transformed hematopoietic stem/progenitor cells with an enhanced or acquired self-renewal capability function as leukemic stem cells. In a variety of solid cancers, stem/progenitor cells could be also targets of carcinogenesis. However, it remains unclear whether disruption of stem cell function directly contributes to cancer initiation. We sought to elucidate the mechanisms of self-renewal in hepatic stem/progenitor cells and the relation between stem cell function and hepatocarcinogenesis. METHODS: Functional analyses of polycomb-group protein Bmi1 and Wnt/beta-catenin, the molecules that are responsible for the self-renewal capability of many types of stem cells, were conducted in c-Kit(-)CD29(+)CD49f(+/low)CD45(-)Ter-119(-) hepatic stem/progenitor cells using retrovirus- or lentivirus-mediated gene transfer. The tumorigenicity of these cells transduced with the indicated retroviruses was also assessed by transplantation into nonobese diabetic/severe combined immunodeficient mice. RESULTS: Forced expression of Bmi1 and constitutively active beta-catenin mutant similarly promoted the self-renewal of hepatic stem/progenitor cells. The transplantation of Bmi1- or beta-catenin-transduced cells clonally expanded from single hepatic stem/progenitor cells produced tumors, which exhibited the histologic features of combined hepatocellular and cholangiocarcinoma. CONCLUSIONS: These observations imply that the dysregulated self-renewal of hepatic stem/progenitor cells serves as an early event in hepatocarcinogenesis, and they highlight the important roles of Bmi1 and the Wnt/beta-catenin pathway in regulating the self-renewal of normal or cancer stem cells in liver.     

狂犬病毒载体的构建与鉴定

目的 构建狂犬病毒载体,为新型疫苗研究提供依据.方法 将狂犬病毒减毒株SAE基因组分段扩增,然后经逐步拼接得到全基因组克隆,并在其中引入转录终止起始元件和多克隆位点获得重组质粒pSAETOPO.将绿色荧光蛋白(EGFP)基因和西尼罗病毒prME基因分别克隆至pSAETOPO,然后将带有这两个基因的狂犬病毒全长cDNA切下并克隆至pcDNA3.1( )载体.将获得的重组质粒pcSAE-GFP和pcSAE-ME转染细胞,分别观察辅助病毒对外源基因表达的影响.结果 获得狂犬病毒载体质粒pcSAE-GFP和pcSAE-ME,经酶切分析和序列测定表明所构建克隆是正确的.pcSAE-GFP及pcSAE-ME转染细胞后,在辅助病毒存在下可表达相应外源基因.结论 构建的狂犬病毒载体质粒可表达外源基因,为进一步获得表达外源基因的重组狂犬病毒奠定了基础.     

携带人miR302和EGFP基因慢病毒表达载体构建

目的构建携带人miR302和增强型绿色荧光蛋白（EGFP）基因的慢病毒表达载体pFUM3GW。方法NheⅠ和NotⅠ双酶切pmiR302ApE以释放miR302,接着补平酶切位点而获连接用miR302;BamHⅠ酶切携带EGFP的慢病毒载体pFUGW,接着补平酶切产物,并去磷酸化而获连接用载体片段,最后使用DNA连接试剂盒（TaKaRa）中的SolutionI将其与连接用miR302连接,连接产物转化,次日挑选单菌落,PCR筛选正向阳性克隆,随后将选定的含有阳性克隆的单菌落摇菌,提取质粒并行酶切鉴定及对插入的miR302测序。所构建载体命名为pFUM3GW。获pFUM3GW后,按Invitrogen公司推荐的标准程序进行慢病毒包装和确认慢病毒是否成功生产;携带miR302和EGFP基因的慢病毒感染鼻咽癌细胞株C666-1、CNE1和5-8F以建立相应病毒感染体系。结果PCR、酶切和测序证实成功构建了pFUM3GW,按标准程序生产的携带miR302和EGFP基因的慢病毒上清高效率感染小鼠胚胎成纤维细胞（MEFs）及鼻咽癌细胞株C666-1、CNE1和5-8F。结论成功构建携带人miR302和EGFP基因的慢病毒表达载体pFUM3GW,为相关后续研究打下了良好基础。     

抗原特异TCR Vα6-pIRES2-EGFP和TCRV β13-pIRES2-EGFP真核表达载体的构建及共转染研究

目的 构建携带弥漫大B型非霍奇金淋巴瘤(DLBL)相关抗原特异性的TCR Vα和TCR Vβ基因片段的真核表达载体TCR Vα-pIRES2.EGFP和TCR Vβ-plRES2-EGFP,将其共同转染Raji和Jurkat细胞.方法 前期研究从1例DLBL患者外周血T细胞中发现克隆性增殖T细胞受体(TCR)Vα6和Vβ13亚家族T细胞,在此基础上利用RT-PCR扩增TCR Va6和TCR Vβ13基因全长序列后,分别将其定向克隆入带有绿色荧光蛋白(EGFP)的真核表达载体pIRES2-EGFP,酶切和核酸序列测定分析方法鉴定重组质粒TCR Vα6-pIRKS2-EGFP和TCR Vβ13-pIRES2-EGFP的正确性;利用核转染技术(nueleofector)将其分别或共同转染Raji细胞和Jurkat细胞,24 h后利用激光共聚焦显微镜观察EGFP的瞬时表达情况,48h后利用实时定量PCR检测TCR Vα6和TCR Vβ13基因的表达情况,Western blot检测EGFP蛋白的表达情况.结果 获得来自DLBL患者的TCR Vα6和TCR Vβ13基因全长序列,酶切分析和核酸序列测定证实TCR Vα6-pIRES2-EGFP和TCR Vβ13-pIRES2-EGFP重组质粒构建正确;转染24 h后,激光共聚焦显微镜下可观察到EGFP的表达,40%以上细胞发出绿色荧光,单独转染和共转染组荧光产生情况相似;实时定量PCR在单独转染和共转染组均町检测到TCR Vα6和TCR Vβ13基因的表达,共转染组2基因的表达水平稍低于单独转染组;Western blot检测在单独转染和共转染组均显示EGFP蛋白的表达,2组的蛋白杂交带强度相似.结论 成功构建了DLBL特异性的TCR Vα6-pIRES2-EGFP和TCR Vβ13-pIRES2-EGFP真核表达质粒,两者可同时转染到细胞中,并实现了体外共表达.