A non-linear model for measuring grapevine leaf thickness by means of red-edge/near-infrared spectral reflectance

Vegetation is a key element of our ecology system. The leaf area and its thickness provide valuable information about the status of our environment. Thus, there is a need for accurate, efficient, practical methodologies to estimate this biochemical parameter. Hyperspectral measurement is a means of quickly assessing leaf parameter in situ. In the past decades, there were lots of work (Boyd et al.) that focused on measurement of leaf area index, but very few on measurement of leaf thickness. In this paper, reflectance of grape leaves was measured over the spectral range of 350–1010 nm. The corresponding thickness of leaves from four grapevine cultivars was also measured as part of seventeen field campaigns undertaken during the summer of 2007. An artificial-intelligence technique, the support vector machine (SVM) model, was introduced to establish the relationship between the leaf thickness and red-edge/near-infrared (NIR) reflectance, with variability examined among individual cultivars as well as at various growth stages. The best wavelengths were variable depending on the grape cultivar and growth stage. The SVM model allows compilation of factors such as cultivar and growth stage with spectral information to yield a superior result.     

HIV－1载体中存在着提高转录和翻译基因的元件

目的研究HIV-1载体中的一些元件如Rev和Tat蛋白对其骨架的转录及外源基因表达水平的影响。方法将HIV-1表达GFP载体（FUGW）单独或分别与Rev蛋白表达质粒（pLP2）、Tat蛋白表达质粒（pcDNA3.1-Tat）,及表达Rev和Tat蛋白的质粒（△8．9）等摩尔共转染人293T细胞后,经实时定量RT-PCR、FACS、荧光显微镜镜检等方法检测,比较其表达量。结果Rev与RRE结合后,载体骨架及外源基因的转录是单独转染FUGW时的3倍,Tat与TAR结合后,则提高其骨架及外源基因的转录近4倍,而Rev和Tat蛋白的协同作用,其转录本则可提高至6倍。FACS和荧光显微镜镜检也显示GFP蛋白表达量明显提高。F-TPO载体（HIV-1载体乳腺特异表达促血小板生成素）与△8．9在小鼠乳腺上皮细胞HC-11共转染和表达,则TPO蛋白的表达量接近pcDNA3．1-TPO载体的8倍。结论HIV-1载体中存在着提高转录和翻译基因的元件,可提高其骨架的转录和外源基因的表达,且该现象并不依赖于细胞类型和外源基因的种类。     

高糖应激致人PRKCD基因过表达内皮细胞模型构建及鉴定

目的:构建高糖应激下人PRKCD基因过表达内皮细胞模型并鉴定。方法:设计含AgeⅠ和NheⅠ酶切位点的PRKCD基因上下游引物,以含PRKCD基因的原始质粒为模版,PCR扩增获得PRKCD全部序列,经AgeⅠ和NheⅠ酶切后与同样酶切后的真核表达载体pDC316-LacZα重组获得穿梭质粒pDC316-PRKCD,经PCR及酶切、基因测序鉴定后,与腺病毒骨架质粒pB-HGlox△E1,3Cre共转染293细胞获得重组腺病毒Ad5-PRKCD,行PCR鉴定并反复纯化扩增后用TCID50法测定病毒滴度。分组培养人脐静脉内皮细胞,转染重组腺病毒后于高糖(25mmol/L)负荷,并设立空载体对照及渗透压对照组,以免疫荧光法检测PKCδ在细胞中的表达。结果:目的基因成功插入穿梭质粒,基因测序结果与GenBank公布序列一致,重组腺病毒Ad5-PRKCD经PCR鉴定及免疫荧光鉴定成功。测得病毒滴度为1.0×1010IU/ml。激光共焦聚观察高糖负荷下,胞内PRKCD翻译产物PKCδ荧光表达强度明显增强,为正常对照组的1.5倍(P0.05),高糖负荷下内皮细胞感染重组腺病毒后PKCδ荧光强度明显增加,浆/核荧光强度比值较高糖组进一步降低了35%(p0.05),提示核转位明显。结论:成功构建了人重组腺病毒Ad5-PRKCD并有效转染人脐静脉内皮细胞,高糖负荷使PKCδ表达上调并发生核转位激活,为筛选稳定表达PKCδ的内皮细胞株及其蛋白复合体奠定了基础。     

Vector fragmentation: Characterizing vector integrity in transfected clones by Southern blotting

The Chinese Hamster Ovary production cell line development process using methotrexate (MTX) amplification is well studied and commonly used for biopharmaceutical processes. However, successful MTX amplification varies from clone to clone and suggested reasons include vector fragmentation during the transfection process and genomic rearrangement of the Chinese Hamster Ovary chromosomes. Here, we elucidated the vector integration patterns of 40 transfected single‐cell clones by Southern blotting and showed that vector fragmentation occurs at a significant level in our experiment. This concurs with MTX amplification studies implying that single‐cell cloning is necessary to ensure a successful amplification process. Truncations at the ends of the integrated vectors were also observed, whereas gross DNA insertions were not detected in our data. This suggests that end deletions are common, whereas insertion events are rare in animal cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.     

Investigation of polyethylenimine‐grafted‐triamcinolone acetonide as nucleus‐targeting gene delivery systems

Background

Nuclear membrane is one of the main barriers in polymer mediated intracellular gene delivery. To improve the transgenic activity and safety of nonviral vector, triamcinolone acetonide (TA) as a nuclear localization signal was conjugated with different molecular weight polyethylenimine (PEI).

Methods

Different molecular weight PEI [600, 1800, 25 000 (25k)] was conjugated with TA to synthesize PEI‐TA by two‐step reaction. Their physicochemical characteristics, in vitro cytotoxicity and transfection efficiency were evaluated. To investigate the difference of transfection efficiency of various molecular weight PEI‐TA, their transfection mechanism was further investigated by confocal microscopy and competition assay. Transgenic expression in vivo was evaluated by injection into hepatic portal vein of mice.

Results

All PEI‐TA could form nanosize polyplexes with DNA and their physicochemical properties resemble each other. Their cytotoxicities were negligible compared to PEI 25k. The order of transfection efficiency was PEI 1800‐TA > PEI 600‐TA > PEI 25k‐TA. A transfection mechanism study displayed that TA could inhibit considerably the transgenic activity of PEI 1800‐TA and PEI 600‐TA, but that of PEI 25k‐TA was not inhibited. It was suggested that PEI 1800‐TA and PEI 600‐TA might translocate into the nucleus. Confocal microscopy investigation verified this suggestion. The data strongly suggested that the transfection efficiency of PEI 1800‐TA in vivo was much higher than that of PEI 25k, which was consistent with the results obtained in vitro.

Conclusions

Low molecular weight PEI‐TA could translocate into the nucleus efficiently. PEI 1800‐TA presented higher transgenic activity and it has a great potential for gene therapy as a nonviral carrier. Copyright © 2010 John Wiley & Sons, Ltd.     

An S/MAR-based L1 retrotransposition cassette mediates sustained levels of insertional mutagenesis without suffering from epigenetic silencing of DNA methylation

L1 is an insertional mutagen that is capable of mediating permanent gene disruption in mammalian genomes. However, currently available L1 retrotransposition vectors exhibit low or unstable transgene expression when expressed in somatic cells and tissues. This restriction limits their potential utility in long-term screening procedures or somatic mutagenesis applications. In this study, we addressed this problem by developing a minicircle, nonviral L1 retrotransposition vector using a scaffold/matrix attachment region (S/MAR) in the vector backbone and evaluated its utility in human cell lines. The S/MAR-based L1 retrotransposition vector provides stable, elevated levels of L1 expression compared to the currently used EBNA1-based L1 vector. In addition, the S/MAR elements effectively mediate sustained levels of L1 retrotransposition in prolonged cell culturing without suffering from epigenetic silencing by DNA methylation or from vector integration problems even in the absence of selection pressure. These findings indicate that the simple inclusion of S/MAR in the vector backbone increased levels of L1 expression and retrotransposition that can be used as an effective tool to generate insertional mutagenesis in large-scale somatic mutagenesis applications in mammalian cells.     

过表达Nanog基因的小鼠骨髓间质干细胞对NF-κB表达的影响

核因子κB(nuclear factor kappa B,NF-κB) 的激活被认为与中枢神经系统变性疾病的进展有关。新近报告Nanog能抑制NF-κB的表达,为验证这一发现,通过限制性内切酶酶切和基因重组的方法,构建携带Nanog基因的重组慢病毒表达载体质粒pNL-Nanog-IRES2-EGFP,经PCR检测以及测序鉴定后,在脂质体介导下与包装质粒HELPER、包膜质粒VSVG共转染293T细胞包装生产慢病毒。所获慢病毒感染小鼠骨髓间充质干细胞 (mMSCs) 后,Western blotting法检测在mMSCs中Nanog 基因的表达,PCR、Western blotting和免疫细胞化学法检测NF-κB基因的表达。结果显示所克隆的Nanog基因测序结果与GenBank报道序列完全一致。构建的慢病毒载体质粒PNL-Nanog-IRES2-EGFP经Sal I和BamH I双酶切后电泳鉴定正确。所获慢病毒感染mMSCs后荧光激发mMSCs可见绿色荧光,Western blotting检测显示Nanog-mMSCs 组表达Nanog,其他两组基本不表达。RT-PCR和Western blotting检测显示Nanog-mMSCs组的NF-κB表达较空载体-mMSCs组及mMSCs组低,有显著性差异。构建携带Nanog基因慢病毒载体并在小鼠骨髓间质干细胞中成功表达,Nanog基因的表达可抑制NF-κB表达,这结果为神经变性疾病的治疗提供了新思路。     

心钠素表达细胞移植降低高血压大鼠的血压和增加其尿量

为探讨心钠素基因转移治疗高血压和慢性心肾功能衰竭等慢性疾病的潜力,首先利用逆转录病毒载体获得可表达和分泌人心钠素的遗传工程细胞,然后将这种细胞植于自发性高血压大鼠SHR的皮下。结果发现,人心钠素遗传工程细胞的移植可使动物血浆中的心钠素浓度在移植后第7天时明显升高。在整个实验期间,虽然实验组动物的血压会随个体发育而逐渐升高,但在实验开始后的42 d内却始终明显低于空载体组,其中第14天血压的差异高达33 mm Hg。在实验开始后的第14天和第21天,实验组动物的尿量也明显增加。以上结果说明,人心钠素遗传工程细胞的皮下移植可明显抑制SHR大鼠血压的上升趋势和改善其泌尿功能,提示该方法具有治疗高血压和慢性心肾功能衰竭等慢性疾病的潜力。