ΦC31整合酶介导猪基因组定点修饰的探讨

高效与特异的基因组定点修饰是基因工程动物研究的前沿与难点.链霉菌噬菌体ΦC31整合酶能介导含attB位点的外源基因定点整合于多种真核生物基因组的假attP位点,可维持外源基因的正常结构及高效表达.本文探讨ΦC31整合酶介导外源基因在猪基因组内定点整合的分子基础.构建含attB位点的报告载体pEGFP-N1-attB,与ΦC31整合酶表达载体pCMV-INT共转染猪肾PK15细胞,G418筛选获得单克隆细胞系.实时荧光定量PCR筛选出单拷贝整合的转基因细胞系.TAIL-PCR鉴定出1个猪基因组假attP位点,位于猪1号染色体,watson链,坐标114220087-114220126,命名为pig-attP-1.测序结果显示,pEGFP-N1-attB在attB位点处断开插入到pig-attP-1.用荧光计测定细胞培养基上清EGFP含量发现,该转基因细胞系EGFP的表达水平是本底的50倍(13500 AU vs.280 AU),表明pig-attP-1是利于外源基因高效表达的\"友好位点\".该研究不仅为实现外源基因在猪基因组内的定点整合提供了新策略,也为创制基因工程猪、建立动物生物反应器等研究注入了新思路.     

不同生育期土壤基质势调控对棉花生长和土壤水盐分布的影响

针对南疆地区水资源短缺和棉田土壤盐碱化问题,研究不同生育期土壤基质势调控对棉花生长和土壤水盐的影响,为棉田节水控盐和高效生产提供理论依据。通过大田膜下滴灌试验,以棉花灌水时期滴头下方20 cm处土壤基质势下限控制水平-50 kPa为对照(CK),在棉花的苗期(A)、苗期+蕾期(B)、苗期+蕾期+花铃期(C)设置3个基于土壤基质势下限的灌溉水平:W₁(-20 kPa)、W₂(-30 kPa)和W₃(-40 kPa),测定棉花生长、地上干物质量、产量和土壤水盐分布等指标。结果表明:不同生育期土壤基质势调控时,株高、叶面积指数和地上干物质量均表现为:WC>WB>WA>CK;不同土壤基质势水平调控时,随着土壤基质势下限的提高,株高、叶面积指数和地上干物质量也随之增加,其中,W₁C和W₂C处理显著高于其他处理。有效铃数、单铃重和衣分等产量构成要素均随着土壤基质势下限的升高而增加。W₁C和W₂C处理棉花的产量基本一致且显著高于其他处理,W₂C的水分利用效率显著高于W₁C处理。不同生育期土壤基质势均控制在-20或-30 kPa可以改善棉花主根区水分状况。各处理在收获期均表现为浅层积盐(0～40 cm),且膜外大于膜内;土壤基质势越高,膜内主根区(0～40 cm)积盐越少,其中W₁C和W₂C较其他处理减少24%。综合考虑高效生产和节水控盐,建议将当地休作期未淋洗棉田灌水时期土壤基质势控制在-30 kPa为宜。     

Abnormalities in neural crest cell migration in laminin alpha5 mutant mice

Although numerous in vitro experiments suggest that extracellular matrix molecules like laminin can influence neural crest migration, little is known about their function in the embryo. Here, we show that laminin alpha5, a gene up-regulated during neural crest induction, is localized in regions of newly formed cranial and trunk neural folds and adjacent neural crest migratory pathways in a manner largely conserved between chick and mouse. In laminin alpha5 mutant mice, neural crest migratory streams appear expanded in width compared to wild type. Conversely, neural folds exposed to laminin alpha5 in vitro show a reduction by half in the number of migratory neural crest cells. During gangliogenesis, laminin alpha5 mutants exhibit defects in condensing cranial sensory and trunk sympathetic ganglia. However, ganglia apparently recover at later stages. These data suggest that the laminin alpha5 subunit functions as a cue that restricts neural crest cells, focusing their migratory pathways and condensation into ganglia. Thus, it is required for proper migration and timely differentiation of some neural crest populations.     

Staphylococcus aureus adherence to nasal epithelial cells in a physiological in vitro model

Summary Nasal carriage of Staphylococcus aureus represents a risk factor for subsequent invasive infections and interpatient transmission of strains. No physiological in vitro model of nasal epithelial cells is available to study both patient- and bacteria-related characteristics and their interaction, leading to adherence and colonization. Starting with tissues from human nasal polyps, a confluent, squamous, nonkeratinized epithelium in collagen-coated 96-well microtiter plates was obtained after 14 d. This in vitro cell-layer was characterized histologically, ultrastructurally, and immunohistochemically and showed features that were indistinguishable from those observed in the squamous nonkeratinized epithelium found in the posterior part of the vestibulum nasi. Adherence experiments were performed with four different ³H-thymidine-labeled Staphylococcus aureus strains. The effect of bacterial inoculum size, temperature of incubation, and incubation medium were studied. The adherence results were found to be reproducible, reliable and sensitive, allowing detection of small quantitative differences in adherence between the Staphylococcus aureus strains. There was no significant difference in adherence at 23° C and 37° C, nor between the incubation medium M199 and phosphate-buffered saline. Plastic adherence could be reduced and standardized with use of siliconized tips and a constant bacterial inoculum volume of 100 μl/well. This physiological and reliable in vitro cell-culture model offers a unique opportunity to study Staphylococcus aureus adherence to squamous, nonkeratinized nasal epithelial cells and both patient and bacterial characteristics involved in this interaction.     

How do plant ecologists use matrix population models?

Matrix projection models are among the most widely used tools in plant ecology. However, the way in which plant ecologists use and interpret these models differs from the way in which they are presented in the broader academic literature. In contrast to calls from earlier reviews, most studies of plant populations are based on < 5 matrices and present simple metrics such as deterministic population growth rates. However, plant ecologists also cautioned against literal interpretation of model predictions. Although academic studies have emphasized testing quantitative model predictions, such forecasts are not the way in which plant ecologists find matrix models to be most useful. Improving forecasting ability would necessitate increased model complexity and longer studies. Therefore, in addition to longer term studies with better links to environmental drivers, priorities for research include critically evaluating relative/comparative uses of matrix models and asking how we can use many short-term studies to understand long-term population dynamics.     

MMP-11 promotes papillary thyroid cell proliferation and invasion via the NF-κB pathway

Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer, and its incidence is on the rise. It has been reported that some matrix metalloproteinases (MMPs) are abnormally expressed in PTC and can be used as diagnostic markers. However, few studies have explored the underlying mechanisms by which MMPs promote tumor progression. In this study, we used microarray analysis to compare the variations of gene expression within the PTC cell populations and their adjacent normal tissues and found that MMP-11 was the most differentially expressed MMP. To investigate the role of MMP-11 in the mediation of thyroid cancer cell development, pEnter-MMP-11 plasmid, and MMP-11 small interfering RNA were applied to up- and downregulate MMP-11 expression of in cultured PTC cell lines K1 and BCPAP. The results suggested that the levels of proliferation and migration of cells transfected with MMP-11 siRNA were significantly reduced, while the levels in MMP-11-plasmid-transfected cells were increased. In terms of the mechanism, experimental data showed that the change in cyclin D1 is consistent with MMP-11 expression, which may explain the changes in proliferation. In addition, Western blot assay was conducted to analyze the p65 and activated (phospho-) p65 protein levels concomitant with MMP-11 adjustments. Variations in intracellular MMP-11 significantly altered the amount of phospho-p65 in thyroid cells, while p65 knockdown did not affect MMP-11 expression. These results suggest that MMP-11 is located upstream of p65 and regulates its activity. Interestingly, the data for the Transwell assay suggested that MMP-11 regulatory migration is also associated with the NF-κB p65 signaling pathway. In conclusion, this report describes the important role of MMP-11 in the regulation of thyroid cell proliferation and migration. Mechanistic studies have shown that cyclin D1 and p65 are important mediators in the processes, which provides a new way to study the mechanism of MMPs promoting the progression of thyroid cancer.     

滇牡丹自然种群数量动态

运用种群静态生命表、存活曲线、生殖力表和Leslie矩阵模型研究了中国西南特有濒危植物滇牡丹(Paeonia delavayi)种群数量动态过程。静态生命表和种群存活曲线表明: 滇牡丹在株龄3-6 a之间经历了较强的环境筛, 其单株生理寿命为15 a左右, 平均周期为8 a, 种群的净增殖率(R₀ = 0.985 7)、内禀增长率(r_m = -0.001 7)和周限增长率(λ = 0.998 3)表明其为衰退型种群; 滇牡丹种群存活表现为台阶型曲线(B1型), 分别在6 a和12 a阶段种群消亡率(K_x)较高。Leslie矩阵模型的模拟结果表明, 在未来30 a内种群数量呈现出下降趋势, 大约下降了50%, 其种群数量靠自身根系的萌蘖和种子繁殖共同维持。野生滇牡丹种群数量下降与其自身繁殖特性有关, 但主要原因是人为采挖和生态环境的破坏。