knock-in法建立Cre重组酶表达鼠系

目的 建立神经特异性表达的Cre重组酶鼠系 ,以便在特定的组织中 ,于某一特定的时间段 ,用条件性基因剔除技术在活体状态下研究基因功能。方法 用knock -in技术 ,以微小管相关蛋白 1a(Mapla)启动子控制Cre重组酶表达。结果 PCR检测结果表明 ,Cre重组酶基因已成功植入该鼠系中。结论 本研究为配合条件性基因剔除技术 。

慢病毒介导的CYP3A11基因knock-down小鼠的建立

目的构建和筛选小鼠P4503A11基因miR RNAi慢病毒载体,建立CYP3A11基因knock-down小鼠。方法利用Ravi Sachidanandam's Lab的在线软件设计沉默小鼠P4503A11基因的miRNA所对应的shRNA序列,PCR扩增后克隆到PCI-GFP-MiR质粒中,其再与慢病毒载体FUW进行重组。将psPAX2和pMD2.G两个载体和FUW-GFP-MiR-shRNA重组慢病毒载体共转染293FT细胞,包装成病毒。用包装好的病毒液感染293FT细胞后,并利用GFP蛋白表达水平进行滴度测定。将上述重组慢病毒载体分别转染FVB/N小鼠肝细胞,转染48h后,检测P4503A11基因mRNA表达水平的变化。选取干扰效果最好的FUW-GFP-MiR-shRNA1重组慢病毒载体进行制备基因knock-down小鼠模型。用显微注射法将浓缩的shRNA1病毒液注射至FVB/N小鼠12细胞期胚胎透明带下。将注射后发育至22细胞期的胚胎移植至假孕受体母鼠,得F0代小鼠。在紫外光照射下利用滤光片观察GFP在小鼠活体内的表达水平。结果DNA测序结果显示3个重组慢病毒载体均与所设计的shRNA序列一致。检测的3个浓缩前慢病毒悬液的滴度均≥106TU/ml,经过高速离心对病毒进行浓缩和纯化,其滴度达到109TU/ml以上。转染FUW-GFP-MiR-shRNA1、2的2组肝细胞的P4503A11基因mRNA表达水平相对于空白对照组均显著下降(P<0.05),而转染阴性对照FUW-GFP-MiR-shRNA-NC的肝细胞P4503A11基因mRNA表达水平相对于空白对照组无明显改变。选用FUW-GFP-MiR-shRNA1慢病毒载体制备的F0代3A11基因knock-down小鼠,在紫外光照射下,阳性小鼠可见较强的荧光。结论构建并筛选出小鼠P4503A11基因有效的靶向miR RNAi慢病毒载体,并成功建立CYP3A11基因knock-down小鼠。

Effects of SIRT1 Gene Knock-Out via Activation of SREBP2 Protein-Mediated PI3K/AKT Signaling on Osteoarthritis in Mice

This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group（6 mice in each group）. In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups： SIRT1^（＋/＋） control group（group A, n=6）; SIRT1^（＋/＋） osteoarthritis group（group B, n=6）; SIRT1^（–/–） control group（group C, n=6）; SIRT1^（–/–） osteoarthritis group（group D, n=6）. HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type Ⅱ collagen proteins. SA-β-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1^（–/–） osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type Ⅱ collagen was destroyed and distributed unevenly. Compared with the SIRT1^（＋/＋） osteoarthritis group and SIRT1^（–/–） control group, SIRT1 protein expression was not obviously changed in the SIRT1^（–/–） osteoarthritis group（P〉0.05）, while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased（P〈0.05） and the levels of AKT and type Ⅱ collagen proteins were significantly decreased（P〈0.05）. SIRT1 gene knock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.

Simulation Research on the Effect of Cooled EGR, Supercharging and Compression Ratio on Downsized SI Engine Knock

Knock in spark-ignition（SI） engines severely limits engine performance and thermal efficiency. The researches on knock of downsized SI engine have mainly focused on structural design, performance optimization and advanced combustion modes, however there is little for simulation study on the effect of cooled exhaust gas recirculation（EGR） combined with downsizing technologies on SI engine performance. On the basis of mean pressure and oscillating pressure during combustion process, the effect of different levels of cooled EGR ratio, supercharging and compression ratio on engine dynamic and knock characteristic is researched with three- dimensional KIVA-3V program coupled with pressure wave equation. The cylinder pressure, combustion temperature, ignition delay timing, combustion duration, maximum mean pressure, and maximum oscillating pressure at different initial conditions are discussed and analyzed to investigate potential approaches to inhibiting engine knock while improving power output. The calculation results of the effect of just cooled EGR on knock characteristic show that appropriate levels of cooled EGR ratio can effectively suppress cylinder high-frequency pressure oscillations without obvious decrease in mean pressure. Analysis of the synergistic effect of cooled EGR, supercharging and compression ratio on knock characteristic indicates that under the condition of high supercharging and compression ratio, several times more cooled EGR ratio than that under the original condition is necessarily utilized to suppress knock occurrence effectively. The proposed method of synergistic effect of cooled EGR and downsizing technologies on knock characteristic, analyzed from the aspects of mean pressure and oscillating pressure, is an effective way to study downsized SI engine knock and provides knock inhibition approaches in practical engineering.

Theoretical Analysis of Noise of Piston Knocking Cylinder Wall in Automotive Engines

Based on the loading conditions of engine, applying difference method to solve the hydrodynamic lubrication equation of piston skirt movement, the force acting on piston skirt and the moment on wrist pin were obtained. A computer program for simulating the piston second order motion was conducted to calculate the lateral motion of the upper part and the bottom part of piston skirts of the engine of automotive model CA1091. From the simulated result, the maximal impacting phase and the maximal impacting region of the piston were obtained. The result can be used for designing engine, diagnosing the noise of piston knocking cylinder wall and explaining many practical fault phenomena in theory.

Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat（CRISPR）/Cas9-mediated gene knock-ins with zinc finger nucleases（ZFNs） and transcription activator-like effector nucleases（TALENs） in bovine and dairy goat fetal fibroblasts. To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin（MSTN） gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2. Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in theGFP-PGK-Neo R plasmid background, including a 5′ and 3′ homologous arm flanking the genes humanized Fat-1（h Fat-1） or enhanced green fluorescent protein（eGFP）. Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and thehFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts. After G418（Geneticin） selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove. The gene knock-in events were screened by PCR across the homologous arms. The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP andhFat-1 gene knock-ins were 13.68 and 0%, respectively. The efficiencies of CRISPR/Cas9-mediated eGFP andhFat-1 gene knock-ins were 77.02 and 79.01%, respectively. The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system. Additionally, thehFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system. The difference of knockin efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP andhFat-1 gene knock-ins were 32.35 and 26.47%, respectively. Theefficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively. The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs. The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines.

运用Mask Pro和Knock Out进行指纹等痕迹物证图像处理的尝试

如何滤除复杂背景,以获取清晰的痕迹物证图像,除应用计算机进行图像处理外,还应借用Photo shop作为平台,尝试运用Mask Pro和Knock Out两款特效插件对指纹图像进行处理,效果会更加理想。

Knock prediction for dual fuel engines by using a simplified combustion model

The present work used a methane-air mixture chemical kinetics scheme consisting of 119 elementary reaction steps and 41 chemical species to develop a simplified combustion model for prediction of the knock in dual fuel engines. Calculated values by the model for natural gas operation showed good agreement with corresponding experimental values over a broad range of operating conditions.

Test Research on the Knock of a Common-Rail Diesel Engine Fueled with Diesel-Methanol Dual-Fuel

Experiments were conducted on a diesel-methanol dual-fuel(DMDF)engine modified by a six-cylinder,turbocharged,inter-cooled diesel engine.According to the number of diesel injection,the experiments are divided to two parts:the single injectionmode and double injectionmode.The results show that,at the double injectionmode,themaximumof pressure rise rate is small and the engine runs smoothly,however,knock still occurswhen the cocombustion ratio(CCR)is big enough.Under knock status,the power density of the block vibration concentrating at some special frequencies rises dramatically,and the special frequency of single injection mode(about 4.1 kHz)is lower than that of double injection mode(7–9 kHz).The cylinder pressure oscillations of knock status are very different fromthe non-knock status.Under knock status,cylinder pressure oscillations become more concentrated and fiercer at some special frequencies,and the same as the block vibration.The special frequency of single injection mode(3–6 kHz)is lower than that of double injection mode(above 9 kHz).

Estimation of Knock Wheel Noise of Rail Transport with an Acoustic Intensity Method

The application of railway wheel noise compensation method is considered. The effect can be obtained by shift a pair of usual parallel rail joints on a pair of offset rail joints, for example, the right rail joint is shifted concerning the left rail joint on the certain distance. The distance is depend on the speed of train and promotes the correlation interconnection between excited acoustical pulses. The intensity wheel knock spectrograms corresponding of different distance between rail joints registered by the microphone are estimated. The spectrograms considerably differed from each other in low frequency band are analyzed. The result of practical using the acoustic noise compensation method is proved.

论海上拖航合同中的Knock for Knock条款

海上拖航合同中的Knock For Knock条款的效力在实践中一直存在争议。本文通过分析《海商法》第162条,结合Knock For Knock条款的性质进行讨论,得出结论《海商法》第162条可以作为订立Knock For Knock条款的依据,但是不能作为该条款在任何情形下均有效的依据。并且通过对Knock For Knock条款效力的进一步探讨,可知海上拖航中的承拖方不得以该条款对抗被拖方提出的由于承拖方的故意或重大过失行为造成其损失的赔偿请求。除此之外,本文将借鉴国外判例,结合我国国情,提出减少Knock For Knock条款实践纠纷的建议。

CRISPR/Cas9 knock-in介导的输卵管特异性表达转基因鸡研究进展

利用动物生物反应器生产重组蛋白是一种具有应用前景的生物技术。鸡输卵管生物反应器是理想的动物生物反应器之一,其优点在于表达的外源蛋白能够分泌到蛋清中,可避免蛋白提取过程中对鸡本身造成伤害,同时蛋清成分简单,便于后期的纯化。目前利用慢病毒结合原始生殖细胞(PGCs)制备转基因鸡被认为是最可行的方法,但因外源基因随机整合且生殖系传递效率较低,使转基因鸡研究受到技术上的限制。而2013年问世的CRISPR/Cas9基因敲入(CRISPR/Cas9 knock-in)技术能够使外源基因精准定向插入基因组特异性位点,这对生产输卵管特异性转基因鸡具有重大意义。文章综述了鸡输卵管反应器的研究进展、CRISPR/Cas9 knock-in技术在输卵管特异性表达转基因鸡研究和鸡育种领域的应用现状,并指出了目前存在的问题和相应的解决办法。

Effect of using borax decahydrate as nanoparticles additive in blends of spirulina biodiesel/diesel on combustion characteristics and knock intensity

In this extensive investigation,the impact of borax decahydrate as a fuel additive in a diesel single-cylinder engine was rigorously examined.Borax decahydrate was introduced at concentrations of 5,15,25 and 35 g in 500 ml of biodiesel,forming five unique fuel mixtures with conventional diesel:90%diesel+10%spirulina biodiesel(SB10),SB10+1 g borax decahydrate(SB10B1),SB10+3 g borax decahydrate(SB10B3),SB10+5 g borax decahydrate(SB10B5)and SB10+7 g borax decahydrate(SB10B7).The investigation encompassed four diverse loading conditions and yielded insightful findings.Notably,at full load,SB10B3 exhibited a higher cylinder peak pressure than diesel,reaching 69.25 bar.Heat release rate profiles demonstrated superior efficiency for SB10 at 50%load,with a cumulative heat release rate of 950 J/°CA,which is lower than the 1050 J/°CA of diesel.Knock intensity(KI)evaluations revealed that,although SB10 and SB10B1 exhibited higher KI than diesel at full load due to elevated peak pressure,SB10B7 showed no knocking across all loads,indicative of reduced in-cylinder combustion.This meticulous numerical analysis emphasizes the potential of borax decahydrate as a catalyst and enhancer,providing valuable insights into the combustion dynamics of these alternative fuel blends and their viability for sustainable and efficient engine performance.In summary,out of all the blends,SB10B3 could be a potential diesel fuel replacement fuel for compression-ignition engines.

Developing a Knock Predictive Criterion in Spark Ignition Engines Fuelled with Gaseous Fuels

Consideration of the chemical reaction activity of the end gas in a spark ignition and operating conditions are combined to predict the onset of knock and associated performance in an engine fuelled with methane.A two-zone predictive combustion model was developed based on an estimate of the efiFective duration of the combustion period and the mass burning rate for any set of operating conditions.The unburned end gas preignition chemical reaction activity is described by a detailed chemical reaction kinetic scheme for methane and air.The variation with time of the value of a formulated dimensionless knock parameter(k)is calculated.It is shown that whenever knocking is encountered,the value of'k' builds up to a sufficiently high value that exceeds a critical value.Under normal operating conditions, the value of'k'remains throughout the whole combustion period at comparatively very low levels. It is shown that the model and the use of this knock criterion'k'produce results that are in good agreement with experiment.

Improvement of engine performance with high compression ratio based on knock suppression using Miller cycle with boost pressure and split injection

In theory,high compression ratio has the potential to improve the thermal efficiency and promote the power output of the SI engine.However,the application of high compression ratio is substantially limited by the knock in practical working process.The objective of this work is to comprehensively investigate the application of high compression ratio on a gasoline engine based on the Miller cycle with boost pressure and split injection.In this work,the specific optimum strategies for CR10 and CR12 were experimentally investigated respectively on a single cylinder DISI engine.It was found that a high level of Miller cycle with a higher boost pressure could be used in CR12 to achieve an effective compression ratio similar to CR10,which could eliminate the knock limits at a high compression ratio and high load.To verify the advantages of the high compression ratio,the fuel economy and power performance of CR10 and CR12 were compared at full and partial loads.The result revealed that,compared with CR10,a similar power performance and a reduced fuel consumption of CR12 at foil load could be achieved by using the strong Miller cycle and split injection.At partial load,the conditions of CR12 had very superior fuel economy and power performance compared to those of CR10.

Overexpression or knock-down of runt-related transcription factor 1 affects BCR-ABL-induced proliferation and migration in vitro and leukemogenesis in vivo in mice

Background Runt-related transcription factor 1 （Runxl） plays a crucial role in hematogenesis and its dysfunction may contribute to leukemogenesis. However, it is not clear whether or not abnormal expression of Runxl will induce leukemia and how the change of Runxl expression level could affect BCR-ABL-induced leukemogenesis. In the present study, we aimed to analyze if abnormal expression of Runxl in BaF3 cells alone would induce teukemogenesis. And we also wanted to know if abnormal expression of Runxl in leukemic cells would affect leukemogenesis. Furthermore, we investigated whether overexpression or knock-down of Runxl in BaF3 cells would induce leukemogenesis. Methods Plasmids containing full-length Runxl cDNA were transduced into BaF3 cells and BaF3-P185wt cells （BCR-ABL transformed BaF3 cells） by electroporation. Plasmids containing a short hairpin RNA of Runxl were transduced into BaF3 cells and BaF3-P185wt cells by electroporation. Runxl expression level was quantified by Western blotting and quantitative real-time PCR. The effects of overexpression or knock-down of Runxl on proliferation, apoptosis and migration of cells were detected in vitro. Then, using MSCV-P185wt-EGFP as a control, we transplanted MSCV-P185wt-Runx1 cells or MSCV-P185wt-shRNA cells into Balb/c mice through tail vein and observed tumorgenesis of the different phenotypes. Results In vitro analysis revealed that overexpression of Runxl in P185wt cells could inhibit cell proliferation and slow down cell migration; while knock-down of Runxl could promote cell proliferation and speed up cell migration. In vivo analysis indicated that mice transplanted with MSCV-P185wt-Runx1 survived longer than controls. In contrast, mice transplanted with MSCV-P185wt-shRNA survived shorter than the control group. Gross pathological analysis revealed that the MSCV-P185wt-Runx1 group had less severe splenomegaly and hepatomegaly compared to the control group, and the MSCV-P185wt-shRNA group had more severe splenomegaly and hepatomegaly. No splenomegaly or hepatomegaly was detected in mice transplanted with MSCV-BaF3-Runxl cells or MSCV-BaF3-shRNA cells. Both the mice of MSCV-BaF3-Runxl group and MSCV-BaF3-shRNA group were healthy with no sign of leukemia for up to three months. Conclusions Overexpression or knock-down of Runxl gene in BaF3 cells alone could not induce leukemogenesis. However, in BaF3-P185wt cells, alteration of Runxl expression could affect BCR-ABL-induced proliferation and migration in vitro and leukemoaenesis in vivo.

RF knock-out extraction in HITFiL

A compact facility for cancer therapy has been designed and is presently under construction. A slow beam extraction system using the RF-Knock Out method and 3rd-order resonance is adopted in the synchrotron of this facility. Eight sextupoles are used, four of them are for correcting the chromaticity and the rest for driving the 3rd-order resonance. In order to save the aperture of vacuum chamber, a 3-magnet bump is adopted during the extraction process. The extraction phase space map and the last 3 turns’ particle trajectory before extraction are given. The matching betatron functions with HEBT （high energy beam transport） are also presented.

Heat Transfer,Knock Modeling and Cyclic Variability in a “Downsized”Spark-Ignition Turbocharged Engine

In the present paper a combined procedure for the quasi-dimensional modelling of heat transfer,combustion and knock phenomena in a “downsized”Spark Ignition two-cylinder turbocharged engine is presented.The procedure is extended to also include the effects consequent the Cyclic Variability.Heat transfer is modelled by means of a Finite Elements model.Combustion simulation is based on a fractal description of the flame front area.Cyclic Variability(CV)is characterized through the introduction of a random variation on a number of parameters controlling the rate of heat release(air/fuel ratio,initial flame kernel duration and radius,laminar flame speed,turbulence intensity).The intensity of the random variation is specified in order to realize a Coefficient Of Variation(COV)of the Indicated Mean Effective Pressure(IMEP)similar to the one measured during an experimental campaign.Moreover,the relative importance of the various concurring effects is established on the overall COV.A kinetic scheme is then solved within the unburned gas zone,characterized by different thermodynamic conditions occurring cycle-by-cycle.In this way,an optimal choice of the “knock-limited”spark advance is effected and compared with experimental data.Finally,the CV effects on the occurrence of individual knocking cycles are assessed and discussed.