这项工作得到了国家自然科学基金（批准号:21576279,20906076）和KRIBB研究计划（KGM2211531）的支持。

竹水合物的制备<ol><li> 在40 mL NaOH溶液中加入5 g竹粉，使其在250 mL烧瓶中达到10％（g / g）终浓度。使用0.05M至0.50M的一系列NaOH溶液，以0.05M的增量。 <ol><li>将混合物在60℃或100℃的水浴中孵育60分钟。在121℃下在高压釜中孵育。 </li></ol></li><li> 孵育后，将混合物在室温下静置4小时。然后，取出上清液。加入100ml淡水并混合。 <ol><li>重复洗涤5-6次，直到上清液的pH达到6.8。使用所得固体进行下一步酶促水解。 </li></ol></li><li>对于水解，使用98％H 2 SO 4调节50mL上述混合物至pH 5.0。然后加入0.5 mL 200张滤纸活性（FPU）/ mL纤维素酶。 注意:纤维素酶与竹子的最终比例每1g竹子要20 FPU。 </li><li>将混合物在振荡器中在50℃下孵育36小时。 注意:水解后，混合物中将残留一些固体。 </li><li>通过以7,690 xg离心5分钟获得透明的水解产物。 </li><li>用配有折射率检测器和光电二极管阵列检测器的高压液相色谱系统对葡萄糖，木糖和其他化学物质进行定量。使用HPX-87H柱（300 mm x 7.8 mm）和流动相为5 mM H 2 SO 4溶液，流速为0.8 mL / min。 </li><li> 对于大量的水解产物，在30L不锈钢罐中混合2kg竹粉和18L 0.25M NaOH。 <ol><li>在121℃下在高压釜中孵育罐60分钟。孵育后，用60升塑料罐中的40升水清洗混合物。重复洗涤5次。 </li><li>用水调整洗涤的竹子总量为20升。使用98％H 2 SO 4将混合物的pH调节至5.0。 </li><li>在配备有机械搅拌器的改进的摇动水浴中将混合物酶水解，以确保混合物分布良好。加入200 mL 200 FPU / mL纤维素酶，并将混合物在50℃孵育36小时。 </li><li>水解后，以4,700 xg离心混合物10分钟。 </li></ol></li></ol> 2.野生型肺炎支原体的 2,3-丁二醇生产<ol><li>使用3升竹水解产物作为介质制备的溶剂。 </li><li>制备含有4g / L玉米浆，2g / L（NH 4 ） 2 SO 4，6g / LK 2 HPO 4,3g / L KH 2 PO 4和0.2g / L MgSO 4的发酵培养基。使用2.5M NaOH将介质的pH调节至中性。使用含有3升高压灭菌发酵培养基的5升生物反应器进行发酵。 </li><li>使用肺炎支原体细胞储存在-80℃的低温冰箱中。 </li><li>对于种子培养物，在37℃下，以200rpm摇动培养含有50mL Luria-Bertani（LB）培养基的250mL烧瓶过夜。使用含有10g / L胰蛋白胨，5g / L酵母提取物和10g / L NaCl的LB培养基。 注意:种子培养的细胞密度应达到OD 2（600nm处的光密度）。 </li><li>接种一瓶50ml的种子培养物在生物反应器中。在pH 6.0和37℃保持培养。使用2升/分钟的空气补充速率和250rpm的搅拌速率，产生微量厌氧条件。 </li><li> 在发酵过程中每2 h收集5 mL样品，并用高压液相色谱法分析，以确定肉汤中的化学物质浓度。 <ol><li>使用MFCS / DA在线监测添加到生物反应器中的碱。 注意:有机酸在发酵过程中产生，a加入NaOH以保持培养物pH稳定。加入的碱的体积代表产生的酸的量。碱添加线高原时，由于碳源耗尽或细胞死亡，该过程完成。 </li></ol></li></ol> 3.由肺炎克雷伯菌的budC突变体产生R-丝瓜素 <ol><li>制备含有4g / L玉米浆，2g / L（NH 4 ） 2 SO 4，3g / L乙酸钠，0.4g / L KCl和0.1g / L MgSO 4的乙偶姻生产的培养基。 </li><li>使用肺炎支原体ΔbudC进行R-丝氨酸生产。重复步骤2.4-2.5。 注意: budC编码2,3-丁二醇脱氢酶。 肺炎克雷伯菌ΔbudC失去2,3-丁二醇脱氢酶活性，产生R-乙酰胆碱代替2,3-丁二醇。 </li><li>在pH 6.0和37℃保持培养。使用4升/分钟的空气补充率和搅拌n速率为450 rpm，有氧条件。 注:补氧量高于2,3-丁二醇生产。 </li><li>按照步骤2.6测定样品。 </li></ol> 4. 肺炎克雷伯菌的budA突变株的2-酮葡萄糖酸生产<ol><li>对于2-酮葡萄糖酸生产，使用与R-组氨酸生产相同的培养基。 </li><li>使用肺炎克雷伯菌ΔbudA生产2-酮葡萄糖酸。重复步骤2.4-2.5。 注意: budA编码α-乙酰乳酸脱羧酶。 肺炎克雷伯菌ΔbudA失去α-乙酰乳酸脱羧酶活性，并以葡萄糖为底物产生2-酮基葡萄糖酸。 注意:2-酮葡萄糖酸合成是酸性条件依赖性过程。开发了2-酮葡萄糖酸生产的两阶段发酵10 ;在第一阶段，将种子培养物接种在生物反应器中。 </li><li&gt;在pH 7.0和37°C保持培养。空气补充速度为4L / min，搅拌速度为500rpm。 注意:在这些条件下，细胞生长非常快（4小时内细胞密度达到OD 7）。然后，发酵进行到第二阶段，在此期间培养物pH降至5.0。不加酸在培养基中产生的有机酸天然导致pH降低。在这些酸性条件下，细胞生长停止，但合成2-酮葡萄糖酸并积累在肉汤中。 </li><li>按照步骤2.6测定样品。使用竹子的水解物作为碳源。 注意:将培养基中的葡萄糖转化为2-酮葡萄糖酸，并将木糖转化为木糖酸。由此得到2-酮基葡萄糖酸和木糖酸的混合物。 </li></ol>

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作者没有什么可以披露的。

肺炎 克雷伯杆菌属于肠杆菌科科克雷伯 菌属 。 肺炎克雷伯病在土壤，植被和水等自然环境中广泛分布14 。本研究中使用的野生型肺炎克雷伯杆菌菌株从土壤中分离，用​​于1,3-丙二醇生产15 。这种物种的肺炎支原体和突变体在不同条件下产生许多化学物质。 培养pH和空气补充是影响肺?...

肺炎克雷伯杆菌是一种在有氧和无氧条件下都能良好生长的细菌。 肺炎支原体是用于生产许多化学物质的重要工业微生物。 1,3-丙二醇是一种有价值的化学物质，主要用作合成聚对苯二甲酸丙二醇酯的单体。聚对苯二甲酸丙二醇酯是一种可生物降解的聚酯，其表现出比1,2-丙二醇，丁二醇或乙二醇1更好的性能。 1,3-丙二醇由肺炎链球菌在氧气限制条件下使用甘油作为底物生产2 。 2,3-丁二醇及其衍生物在塑料，溶剂生产和合成橡胶领域有应用，有潜力被用作生物燃料3 。以葡萄糖为底物，2,3-丁二醇是野生型菌株4的主要代谢物。 2,3-丁二醇是合成的从丙酮酸。首先，两分子丙酮酸缩合得到α-乙酰乳酸;该反应由α-乙酰乳酸合成酶催化。然后通过α-乙酰乳酸脱羧酶将α-乙酰乳酸转变成乙偶姻。当丁二醇脱氢酶催化时，β-维生素可进一步还原成2,3-丁二醇。已经探索了适用于肺炎克雷伯菌的有效基因替代方法，并且已经构建了许多突变体5,6,7 。失去其2,3-丁二醇脱氢酶活性的budC突变体在培养液中积累高水平的乙偶姻。乙酰头痛被用作增加食物味道的添加剂。当编码α-乙酰乳酸脱羧酶的budA被突变时，2-ketogluconic acid积聚在肉汤中。 2-酮基葡萄糖酸用于合成异抗坏血酸（异抗坏血酸），用于食品工业的抗氧化剂9 。 2-酮葡萄糖酸是葡萄糖氧化途径的中间体;在该通路中，位于周质空间中，葡萄糖被氧化成葡萄糖酸，然后进一步氧化成2-酮基葡萄糖酸。葡萄糖酸和在周质中产生的2-酮葡萄糖酸可以转运到细胞质进一步代谢。 2-酮基葡萄糖酸的积累取决于酸性条件，更高的空气补充有利于2-酮葡萄糖酸生产10 。葡萄糖酸脱氢酶，由gad编码， 催化葡萄糖酸转化为2-酮聚糖酸。 肺炎克雷伯氏菌的gad突变体产生高水平的葡萄糖酸而不是2-酮葡萄糖酸，并且该过程也依赖于酸性条件。葡萄糖酸是一种大量有机酸，用作添加剂以增加水泥1的性能1。葡萄糖氧化成葡萄糖酸由葡萄糖脱氢酶催化。木糖也是葡萄糖脱氢酶的合适底物。当木糖用作底物时， 肺炎克雷伯菌产生木糖酸12 。 使用生物质作为原料的化学生产是生物技术的热门话题13 。生物质的主要成分是纤维素，半纤维素和木质素。然而，这些大分子化合物不能被大多数微生物（包括肺炎克雷伯菌 ）直接分解代谢。生物质中的纤维素和半纤维素必须水解成葡萄糖和木糖，然后可以被微生物使用。在木质纤维素中木质素的存在产生防止酶的生物质水解的保护屏障。因此，消除木质素和半纤维素并降低纤维素的结晶度的预处理过程总是在通过麦克风的生物量利用期间进行roorganisms。已经开发了许多预处理方法:酸，碱，氨和蒸汽预处理是常见的。 竹子在热带和亚热带地区丰富，是重要的生物质资源。本文介绍了竹水解产物的制备和竹水解产物的化学生产

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">autoclave</td>
			<td style="width:136px;">SANYO</td>
			<td align="right" style="width:119px;">3780</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">bioreactor</td>
			<td style="width:136px;">Sartorius stedim biotech</td>
			<td style="width:119px;">Bostat Aplus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">agitator</td>
			<td style="width:136px;">IKA</td>
			<td style="width:119px;">RW 20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water bath shaker</td>
			<td style="width:136px;">Zhicheng</td>
			<td style="width:119px;">ZWY-110X50</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">high performance liquid chromatograph system</td>
			<td style="width:136px;">Shimadzu Corp</td>
			<td style="width:119px;">20AVP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">centrifuge</td>
			<td style="width:136px;">Hitachi</td>
			<td style="width:119px;">CR22G III</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bamboo powder</td>
			<td style="width:136px;">purchased from Zhejiang Province, China</td>
			<td style="width:119px;"></td>
			<td>mesh number, 50</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">cellulase</td>
			<td style="width:136px;">Youtell Biochemical, Shandong, China</td>
			<td style="width:119px;"></td>
			<td>200 PFU/ml</td>
		</tr>
	</tbody>
</table>

production of chemicals by klebsiella pneumoniae using bamboo hydrolysate as feedstock

竹是重要的生物质，竹科水解产物被克雷伯杆菌用作化学生产的原料。这里，用NaOH预处理竹粉，洗涤至中性pH。将纤维素酶加入预处理的竹粉中以产生含有30g / L葡萄糖和15g / L木糖的水解产物，并用作碳源以制备用于化学生产的培养基。当在微需氧条件下培养时，野生型肺炎克雷伯菌产生12.7g / L 2,3-丁二醇。在需氧条件下， 肺炎支原体 芽胞芽孢杆菌突变体产生了13.0g / L的R-丝氨酸。在肺炎链球菌的budA突变体的两阶段，pH控制的发酵中补充高空气产生了25.5g / L的2-酮基葡萄糖酸和13.6g / L的木糖酸的混合物。在发酵的第一阶段，将培养物保持在中性pH值;细胞生长后，发酵n进入第二阶段，在此阶段，文化被允许变成酸性。

在这个方案中，竹子是用碱预处理的。在图1和图 2中确定了最佳孵育参数 - 温度为121℃和0.25M NaOH。将预处理的竹子酶水解，测定水解产物中得到的葡萄糖和木糖浓度。更高的温度有利于糖的生产，所以选择121°C作为最佳温度。在水解产物中产生的葡萄糖和木糖在NaOH浓度范围内在0.05-0.25Ma范围内增加。NaOH浓度的进一步增加对糖的产生?...

Watch this Scientific Journal Video about Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock at JoVE.com

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

竹粉用NaOH预处理，酶水解。竹纤维素水解产物被用作2,3-丁二醇，β-乙酰胆碱，2-酮基葡萄糖酸和肺炎克雷伯菌产生的木糖酸的原料。

化学品生产<em&gt;肺炎克雷伯菌</em使用竹水合物作为原料

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was ...

production-chemicals-klebsiella-pneumoniae-using-bamboo-hydrolysate

Research

JoVE Journal

Bioengineering

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

8.9K 次观看.  Chinese Academy of Sciences. 竹粉用NaOH预处理，酶水解。竹纤维素水解产物被用作2,3-丁二醇，β-乙酰胆碱，2-酮基葡萄糖酸和肺炎克雷伯菌产生的木糖酸的原料。 

视频: Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

生产尺寸控制肿瘤细胞球体采用微孔板大数

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

科研

生物工程

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

烟草中瞬时表达的蛋白作为一个案例研究:复杂系统采用的实验方法设计的表征

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

生产和目标单价量子点

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

液体中脉冲激光烧蚀制备金属纳米粒子:研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

利用自动 Microbioreactor 技术优化异种蛋白质生产的通用协议

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

CRISPR/Cas9 复合原注射液生产基因工程金黄叙利亚仓鼠

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

含脂脂的微流体生产-温度敏感脂质体

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

腺病毒生产的有效方法

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

化学品生产

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