Name: glutamine flux imaging using genetically encoded sensors
Uploaded: 2014-07-31T13:00:00
Description: Watch this Scientific Journal Video about Glutamine Flux Imaging Using Genetically Encoded Sensors at JoVE.com

这项工作是由美国国立卫生研究院授予1R21NS064412，NSF资助1052048和Jeffress纪念信托基金资助的J-908的支持。

1，样品制备注：在许多情况下灌注实验洗去细胞的显著部分，它可以成为一个令人沮丧的问题。虽然没有必要对所有的细胞系，涂层覆盖玻璃表面用聚-L-赖氨酸（添加0.01％的溶液1.0 ml/25厘米2的表面，孵育&gt; 5分钟，用细胞培养级水洗涤两次，干燥生物安全柜）提高细胞的粘附。同时，要注意使用的细胞系的生物安全水平（BSL）的，并按照批准地方环境健康和安全办?...

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成像实验的成功取决于几个关键因素。其中一个因素是传感器的使用，如上面所讨论的亲和力。在感兴趣的亚细胞区室中衬底的绝对浓度，但是，常常是未知的。因此，我们建议您尝试多种传感器与交错的亲和力，以找到一个所需的实验条件下效果最好。例如，在我们的情况下，我们转染的COS7细胞中谷氨酰胺传感器，1.5μ，100μ，2分，和8米（ 图3和数据未显示）。 <p class="jove_conten...

由于技术的显着进步，使考试的转录组和蛋白质组在细胞水平上的，它现在已经很清楚，生物化学和由此产生的通量代谢物和离子是高度细胞类型特异性。例如，在哺乳动物的肝脏，顺序谷氨酰胺降解和合成是同时进行由门静脉周围的细胞和静脉周围的细胞分别，同时消耗过量的氨在后者的1-3喂养铵到尿素循环在前者的细胞类型。在某些情况下，显著生化异质性被检测，即使在一个单一的“小区类型”4，5。除了 ​​这样的空间特异性，代谢物和离子的细胞水平是高度动态的（ 例如，信号分子，如Ca 2 +，环 ​​核苷酸）。代谢物和离子的时空模式往往能起到关键作用的信号转导。 Monitoring代谢物和离子的细胞动力学，但是，提出了独特的挑战。在许多情况下，其浓度的变化是快速和短暂的，通过信号转导分子如Ca 2 +，内〜20毫秒的树突棘6，该衰变的情况举例说明。此外，内部和之间的细胞的生化途径的分隔使得难以用萃取和柱色谱/质谱技术定量代谢物和离子的动力学。 遗传编码的传感器，用于生物分子现在广泛由于高时空分辨率，允许实验者研究短命和/或隔离的分子动力学使用（7评论，8）。这些基因编码的传感器大致可以分为两大类;基于强度的传感器和ratiometic传感器。强度为基础的传感器通常由一个结合结构域和一个感orescent蛋白（FPS），以及溶质结合的结合结构域改变的荧光强度。 Ratiometic传感器，在另一方面，往往采取的促进共振能量转移（FRET）2的FP充当FRET对的优势。这些传感器包括一个结合结构域和两个FP的，与溶质结合诱导在2的FP之间的FRET效率的变化。大量的传感器，用于生物学上重要的代谢物和离子已被开发，在过去十年中8,9。 其中由该基因编码的传感器所提供的令人兴奋的可能性是他们在膜转运过程的高分辨率分析，这在以前是不容易察觉在细胞水平上的使用。遗传编码的传感器便利的运输机制，如底物特异性和pH依赖性10，11中的分析。此外，在同遗传水库组合OURCES如RNA干扰的库构建的模式生物，它现在可以进行全基​​因组搜索使用基因编码的传感器新颖的运输过程。事实上，利用基因编码的传感器导致的以前未知转运多例12，13的发现。 最近，我们实验室已经开发出一系列基于FRET的传感器谷氨酰胺。我们已经证明，细胞谷氨酰胺水平可以使用这样的FRET谷氨酰胺传感器10被可视化。这些传感器由在C-末端glnH的（ 图1）一个FRET供体（mTFP1）插入到细菌谷氨酰胺结合蛋白glnH和FRET受体（金星）的。这些传感器中的FRET效率下降时的谷氨酰胺结合，导致受体/供体的强度比的降低。谷氨酰胺转运过程的精细调节是重要的生物过程，如neurotransmission 14,15和尿素循环的维持在肝脏1，16，17。 在这里，我们将展示分析运输活动与FRET传感器谷氨酰胺，使用宽场荧光显微镜下建立的方法。这里显示的实验的目的是检测在单个细胞中转运活动，并检查一个瞬时表达转运蛋白的底物特异性。

<table border="0" cellpadding="0" cellspacing="0" width="1129">
	<tbody>
		<tr>
			<td>Inverted fluorescent microscope</td>
			<td>Olympus</td>
			<td>IX81F-3-5</td>
			<td>An equivalent inverted fluorescent microscope from other suppliers would also appropriate.</td>
		</tr>
		<tr>
			<td>Exitation filter (mTFP1)</td>
			<td>Omega Optical</td>
			<td>3RD450-460</td>
			<td>band width 450-460 nm</td>
		</tr>
		<tr>
			<td>Exitation filter (Venus)</td>
			<td>Chroma</td>
			<td>ET500/20</td>
			<td>band width 490-510 nm</td>
		</tr>
		<tr>
			<td>Emission filter (mTFP1)</td>
			<td>Chroma</td>
			<td>HQ495/30m</td>
			<td>band width 475-505nm</td>
		</tr>
		<tr>
			<td>Emission filter (Venus)</td>
			<td>Chroma</td>
			<td>ET535/30m</td>
			<td>band width 520-550nm</td>
		</tr>
		<tr>
			<td>Dichroic mirror (FRET channels)</td>
			<td>Chroma</td>
			<td>470dcxr</td>
			<td>long pass, 470nm cut off</td>
		</tr>
		<tr>
			<td>Dichroic mirror (YFP channel)</td>
			<td>Chroma</td>
			<td>89002bs</td>
			<td>passes 445-490nm, 510-560nm, 590-680nm</td>
		</tr>
		<tr>
			<td>mCherry filter set</td>
			<td>Chroma</td>
			<td>49008</td>
			<td>excitation 540-580nm, long pass dichroic with 585nm cut off, emission filter 595-695nm</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Olympus</td>
			<td>U-LH100L-3-5</td>
			<td>LED- or halogen light source that produces stable light intensity, mercury lamps are not recommended</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Qimaging</td>
			<td>Rolera-MGi EMCCD</td>
		</tr>
		<tr>
			<td>apochromatic fluorescence objective</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>perfusion system, ValveBank II</td>
			<td>AutoMate Scientific</td>
			<td>[01-08]</td>
			<td>Other perfusion systems that allow fast solution exchange would also work</td>
		</tr>
		<tr>
			<td>Laminar-flow chambers</td>
			<td>C&#38;L Instruments</td>
			<td>VC-MPC-TW</td>
			<td>Other larminar-flow chambers would also work</td>
		</tr>
		<tr>
			<td>Chambered slide</td>
			<td>Lab-Tek</td>
			<td>154534</td>
			<td>For open-chamber experiments</td>
		</tr>
		<tr>
			<td>perfusion pump</td>
			<td>Thermo Scientific</td>
			<td>74-046-12131</td>
			<td>For open-chamber experiments</td>
		</tr>
		<tr>
			<td>Software supporting ratiometric measurements</td>
			<td>Intellegenent Imaging Innovation</td>
			<td>Slidebook 5.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow biosafety cabinet</td>
			<td>ESCO</td>
			<td>LA-3A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>13-255-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s MEM (DMEM)</td>
			<td>Hyclone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cosmic Calf Serum</td>
			<td>Hyclone</td>
			<td>SH3008703</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum-free medium for transfection (OPTI-MEM I)</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td>Used with Lipofectamine 2000</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution</td>
			<td>sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>25mm circular glass cover slips</td>
			<td>Thermo Scientific</td>
			<td>12-545-102</td>
			<td>in case VC-MPC-TW is used</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668027</td>
			<td>Other transfection reagents can also be used</td>
		</tr>
		<tr>
			<td>Solution A (Hank&#39;s buffer)</td>
			<td>-</td>
			<td>-</td>
			<td>9.7g HANK salt (Sigma H1387), 0.35g NaHCO3, 5.96g HEPES to 1L, pH adjusted to 7.35 with NaOH</td>
		</tr>
		<tr>
			<td>Solution B</td>
			<td></td>
			<td></td>
			<td>Solution A + 0.04mM Gln</td>
		</tr>
		<tr>
			<td>Solution C</td>
			<td></td>
			<td></td>
			<td>Solution A + 0.2mM Gln</td>
		</tr>
		<tr>
			<td>Solution D</td>
			<td></td>
			<td></td>
			<td>Solution A + 1mM Gln</td>
		</tr>
		<tr>
			<td>Solution E</td>
			<td></td>
			<td></td>
			<td>Solution A + 5mM Gln</td>
		</tr>
		<tr>
			<td>Solution F</td>
			<td></td>
			<td></td>
			<td>Solution A + 5mM Ala</td>
		</tr>
	</tbody>
</table>

glutamine flux imaging using genetically encoded sensors

遗传编码的传感器允许生物分子的亚细胞分辨率的实时监控。一个巨大的品种，例如传感器，生物分子在过去15年中面世，其中一些成为了经常被用在许多实验室不可缺少的工具。 其中一个基因编码传感器的令人兴奋的应用之一是在调查细胞转运过程中使用这些传感器。转运如动力学和底物特异性的属性可以在细胞水平进行调查，为运输活动的细胞类型特异性分析的可能性。在这篇文章中，我们将演示如何转运动力学可以用基因编码的谷氨酰胺传感器为例进行观察。实验设计，实验设置的技术细节，和考虑后的实验分析将进行讨论。

典型时程实验示于图2，在这些实验中，FRET谷氨酰胺传感器为8毫米（FLIPQTV3.0_8m， 图2A和2B）和100μM（FlipQTV3.0_100μC和D）的亲和性被共表达以强制性氨基酸器ASCT2 18 COS7细胞10。谷氨酰胺流入被检测作为给体（mTFP1）和受体（金星）（ 图2A和2C）之间的荧光强度比的变化。谷氨酰胺在另一衬底（丙氨酸）的存在下的流出也?...

Watch this Scientific Journal Video about Glutamine Flux Imaging Using Genetically Encoded Sensors at JoVE.com

Glutamine Flux Imaging Using Genetically Encoded Sensors

本文将展示如何利用FRET监测谷氨酰胺动态活细胞。遗传编码的传感器允许生物分子的亚细胞分辨率的实时监控。实验设计，实验设置的技术细节，并考虑后期的实验分析将用于基因编码的谷氨酰胺的传感器进行讨论。

谷氨酰胺通量成像使用基因编码的传感器

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

Research

JoVE Journal

Bioengineering

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Bacterial Detection & Identification Using Electrochemical Sensors

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

科研

生物工程

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Flying Insect Detection and Classification with Inexpensive Sensors

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An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

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To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

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The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

多显微镜下的血管生成术在基因修饰的 3 d-PLGA/nHAp 支架上进行颅骨临界骨缺损修复

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

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

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. ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

应用交互式、患者特异性模型对神经纤维束进行深部脑刺激治疗的靶向性

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...