“一箭双星法”构建Flt3转基因细胞株

目的构建稳定表达Flt3分子的转基因细胞株。方法将Flt3基因重组入逆转录病毒载体pEGZ-term,通过293T细胞的包装获得具有感染能力的完整重组病毒载体,进而采用“一箭双星法”即同时感染BaF和L929细胞,构建Flt3转基因细胞株。结果成功获得稳定表达Flt3的转基因细胞株BaF/Flt3和L/Flt3。结论采用“一箭双星法”成功构建Flt3转基因细胞株,为以Flt3为靶分子的研究奠定了物质基础,也为类似的高难度的转基因细胞株的构建提供了有价值的经验。

转MSTN干扰载体细胞株的获得及外源基因整合情况的研究

转基因细胞株的建立能够为转基因体细胞克隆技术奠定重要基础。该实验利用MSTN干扰载体,转染鲁西黄牛胎儿成纤维细胞,获得5个相应的转基因单克隆细胞株。采用Realtime PCR和高效热不对称互交式PCR(hiTAIL-PCR)技术检测细胞克隆中MSTN表达载体的拷贝数及其在牛基因组中的整合位点。结果表明,荧光定量PCR有效检测到5个细胞克隆中质粒的拷贝数分别为2.26±0.32、1.52±0.25、25.68±1.02、8.43±0.73和6.72±0.10。hiTAIL-PCR对整合位点的检测结果表明,质粒片段在插入到基因组的过程中进行了重组,其与基因组的结点处有2或4个共同的碱基序列。该研究探索MSTN干扰载体在牛胎儿成纤维细胞中的整合机制,以期获得遗传背景清楚的转基因细胞作为体细胞核移植的重要材料,为高产转基因肉牛新品种的培育提供重要的理论和实验基础。

一种检测线粒体核酸酶靶向剪切活性的新方法

旨在建立一种简便检测线粒体DNA(mt DNA)核酸酶靶向剪切活性的方法。利用转基因技术,将一段含有两个靶向目标序列(T1、T2)的线粒体DNA序列随机整合到宿主基因组中,通过实时荧光定量PCR筛选单拷贝或低拷贝的单克隆转基因细胞株。将含有T1、T2的CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9质粒分别瞬时转染到所选细胞株中,靶向剪切核基因组,在靶向目标序列处造成DNA双链断裂,引发非同源末端连接修复机制,引入插入或缺失突变。观察测序峰图,证明两个靶向目标序列T1、T2均有剪切效率,且T1高于T2。建立了一种高效快速检测线粒体核酸酶靶向剪切活性的新方法。

Screening of the Metastasis-Associated Genes by Gene Chip in High Metastatic Human Ovarian Cancer Cell Lines

Affymetrix U133A oligonucleotide microarrays were used to study the differences of gene expressions between high （H） metastatic ovarian cancer cell line, HO-8910PM, and normal ovarian tissues （C）. Bioinformatics was used to identify their chromosomal localizations. A total of 1,237 genes were found to have a difference in expression levels more than eight times. Among them 597 were upregulated [Signal Log Ratio （SLR） ≥3], and 640 genes were downregulated （SLR≤-3）. Except one gene, whose location was unknown, all these genes were randomly distributed on all the chromosomes. However, chromosome 1 contained the most differentially expressed genes （115 genes, 9.3%）, followed by chromosome 2 （94 genes, 7.6%）, chromosome 12 （88 genes, 7.1%）, chromosome 11 （76 genes, 6.1%）, chromosomes X （71 genes, 5.7%）, and chromosomes l7 （69 genes, 5.6%）. These genes were localized on short-arm of chromosome （q）, which had 805 （65.1%） genes, and the short arms of No.13, 14, 15, 21, and 22 chromosomes were the only parts of the chromosomes where the differentially expressed genes were localized. Functional classification showed that most of the genes （306 genes, 24.7%） belonged to the enzymes and their regulator groups. The subsequent group was the nucleic acid binding genes （144 genes, 11.6%）. The rest of the top two groups were signal transduction genes （137 genes, 11.1%） and proteins binding genes （116 genes, 9.4%）. These comprised 56.8% of all the differentially expressed genes. There were also 207 genes whose functions were unknown （16.7 %）. Therefore it was concluded that differentially expressed genes in high metastatic ovarian cancer cell were supposed to be randomly distributed across the genome, but the majority were found on chromosomes 1, 2, 12, 11, 17, and X. Abnormality in four groups of genes, including in enzyme and its regulator, nucleic acid binding, signal transduction and protein binding associated genes, might play important roles in ovarian cancer metastasis. Those genes need to be further studied.

Down-regulation of miR-622 in gastric cancer promotes cellular invasion and tumor metastasis by targeting ING1 gene

AIM:To evaluate the biological and clinical characteristics of miR-622 in gastric cancer. METHODS:We analyzed the expression of miR-622 in 57 pair matched gastric neoplastic and adjacent non-neoplastic tissues by quantitative real-time polymerase chain reaction. Functional analysis of miR-622 expression was assessed in vitro in gastric cancer cell lines with miR-622 precursor and inhibitor. The roles of miR-622 in tumorigenesis and tumor metastasis were analyzed using a stable miR-622 expression plasmid in nude mice. A luciferase reporter assay was used to assess the effect of miR-622 on inhibitor of growth family,member 1 (ING1) expression. RESULTS:Expression of miR-622 was down-regulated in gastric cancer. MiR-622 was found involved in differentia-tion and lymphatic metastasis in human gastric cancer. Ectopic expression of miR-622 promoted invasion,tumorigenesis and metastasis of gastric cancer cells both in vitro and in vivo. ING1 is a direct target of miR-622. CONCLUSION:These findings help clarify the molecular mechanisms involved in gastric cancer metastasis and indicate that miR-622 modulation may be a bona fide treatment of gastric cancer.

Expression of RECK gene in the osteosarcoma cell line and its correlation with MMP-2

Objective:To detect the expression of RECK gene in the highly and low metastatic cell sublines of human osteosarcoma cell HOS and explore its possible roles on the occurrence and metastasis of osteosarcoma.Methods:RT-PCR, gelatin zymography, and matrigel invasion assay were respectively used to evaluate the endogenous expression of RECK mRNA, MMP-2 activation ratio and invasive capacity in the two osteosarcoma cell sublines.Results:The highly metastatic cell group expressed significantly lower mRNA level of RECK than the low metastatic cell group(P < 0.05), but showed higher MMP-2 activation ratio and invasive capacity(P < 0.05 and P < 0.01, respectively).Conclusion:The abnormal low expression of RECK may participate in osteosarcoma invasion and metastasis, and may be a new therapeutic target for osteosarcoma.

靶细胞表面HCV受体分子排列顺序对病毒入侵影响的研究

目的通过在小鼠肝癌细胞表面串联表达HCV受体分子,在二维结构的基础上研究HCV受体分子的排列顺序对病毒入侵的影响,为研究HCV的早期感染机制奠定基础。方法将已构建的慢病毒表达载体p CDHh LDLR-h SR-BⅠ-h CD81-GFP、p CDH-h LDLR-h CD81-h SR-BⅠ和p CDH-h CLDN-1-h OCLN-DsRed与包装质粒共转染包装细胞293FT,包装慢病毒,用收集浓缩的慢病毒攻击小鼠肝癌细胞系Hepa1-6,抗生素G418加压筛选得到串联表达人紧密链接区域分子CLDN-1和OCLN的转基因细胞株h CLDN-1-OCLN/Hepa1-6(CO/Hepa1-6);重组慢病毒p CDHh LDLR-h SR-BⅠ-h CD81-GFP和p CDH-h LDLR-h CD81-h SR-BⅠ攻击转基因细胞株CO/Hepa1-6,通过嘌罗霉素puro和G418双抗加压及流式细胞术,最终筛选获得5种分子h LDLR、h SR-BⅠ、h CD81、h CLDN-1和h OCLN共表达且具有不同受体排列顺序的转基因人源化小鼠肝癌细胞株LSCCO/Hepa1-6和LCSCO/Hepa1-6;利用HCV阳性血清直接感染方法分析LSCCO/Hepa1-6和LCSCO/Hepa1-6对HCV的易感性,研究HCV受体分子的排列顺序对病毒入侵的影响。结果两种细胞株LSCCO/Hepa1-6和LCSCO/Hepa1-6对HCV结合均有增加,但LSCCO/Hepa1-6细胞株细胞表面的HCV受体排列顺序更有利于HCV入侵。结论在二维结构的基础上,HCV在感染早期要优先与B族Ⅰ型清道夫受体SR-BⅠ作用。

Overexpression of OsDPR, a novel rice gene highly expressed under iron deficiency, suppresses plant growth

Preliminary microarray analysis of cDNA from rice roots revealed an up-regulated transcript that was highly expressed in a five-day iron deficiency treatment. The entire sequence of this gene was determined by bioinformatics analysis. There were no proteins with significant levels of similarity detected in public databases. This novel gene with unknown biological function was designated as OsDPR (dwarf phenotype-related gene). We constructed a stable plant expression vector pCAM- BIA1302-OsDPR::GFP and produced transgenic tobacco plants. The phenotypes suggested that OsDPR restrained the growth of transformed plants. To understand the mechanisms of this suppression effect, cell size and number were compared between transformants and wild-type plants. The cell proliferation rate was lower in OsDPR transgenic BY-2 cells than in wild-type cells, but OsDPR expression did not affect cell size. Moreover, the cell division-related gene CyclinD2.1, which is involved in plant growth, was down-regulated in transgenic tobacco plants. These findings suggested that the novel iron-regulated gene OsDPR is responsible for the nanism phenotype of transgenic seedlings because of the inhibition of plant cell proliferation.