作者感谢格雷松巴特勒和玛格丽特-巴雷特帮助进行实验, 朱迪石头的建议谷物均匀化, 和林恩汉努姆征求意见的荧光。这项工作得到了国家科学基金会 (IOB 0443676) 的支持, 由国立卫生研究院国立医学研究院颁发的机构发展奖 (P20GM0103423), 以及赠款来自科尔比学院自然科学分部。

1. 效应器和报告基因构造的制备
<ol><li>构建使用本构启动子 (如玉米泛素, UBI) 的效应器构造, 以从所需的开放阅读框架中驱动表达式。例如, 构造一个包含UBI::TaABF1的质粒, 以驱动 TaABF15的强本构表达式。</li>
	<li>生成包括要测量其活动的启动器的报告器结构, 放置在打开的阅读框架的上游, 如GUS。例如, 构造一个包含HVA1::GUS的质粒, 用于测量从HVA1启动子2,5的表达式。</li>
	<li>生成内部控件构造 (例如UBI::荧光素酶)。</li>
	<li>使用硅胶绑定列协议 (参见材料表), 为每个必需的基因构造准备高质量的质粒。使用紫外光谱 (参见材料表), 仔细测量每种质粒制备的浓度。</li>
</ol>2. 为轰炸准备大麦谷物
<ol><li>设置电子表格 (请参见图 1 ), 指出将被轰炸的质粒、适当的激素治疗以及将作为实验一部分的每种治疗的其他相关信息。</li>
	<li>使用切割板和无菌剃刀刀片, 从喜马拉雅大麦的谷物中切下胚胎 (试验中包括的每种治疗 40)。排除变色或大大小于平均值的谷物。</li>
	<li>将所有的 embryoless 颗粒放入50毫升管中。加入40毫升的水和岩石10分钟。</li>
	<li>仔细倒入水 (以避免失去谷物), 并添加40毫升10% 漂白剂。摇滚20分钟。</li>
	<li>倒出漂白剂, 加入40毫升的无菌水。摇滚5分钟。重复这种无菌水洗四次。</li>
	<li>添加40毫升的饮用溶液 (20 毫米琥珀酸钠, 20 毫米氯化钙, pH 5.0) 和 40 ul 的氯霉素库存溶液 (10 毫克/毫升)。</li>
	<li>将大部分的饮用溶液 (与氯霉素) 从管子转移到蛭石板 (参见材料表)。然后将谷物转移到蛭石板上湿滤纸的表面。均匀地分散谷物。倒入足够的饮用溶液, 使谷物部分 (但不完全) 淹没。把盖子放在蛭石板的顶部。</li>
	<li>用萤光灯孵化24摄氏度的 embryoless 颗粒, 大约48小时。必要时添加其他饮用解决方案。</li>
	<li>打开一个90毫米塑料培养皿和倒入20毫升的饮用溶液到盘子本身和20毫升进入倒盖。添加 20 ul 的氯霉素 (10 毫克/毫升)。用酒精浸泡两对细点钳。</li>
	<li>将一些 embryoless 谷物放入培养皿的盖子中。使用镊子, 轻轻地去除每粒谷物 (透明) 的种子大衣。从谷物的平滑面除去种子外衣时, 不要损害底层 (糊粉层) 细胞。将去皮的谷物转移到含有饮用溶液的培养皿中。</li>
	<li>剥去所有谷物的种子后, 把它们放回蛭石板上。将去皮的 embryoless 颗粒孵化24摄氏度, 16-20 小时。</li>
	<li>就在使用谷物进行轰炸之前, 通过在培养皿中的滤纸之间摇动来去除表面水分。</li>
</ol>3. 用于轰击的质粒 DNA 的制备
<ol><li>标记一个1.5 毫升管为每个治疗, 将包括在轰炸实验。对每个管添加2.5 µg 的UBI::荧光素酶内部控制质粒, 2.5 µg GUS报告质粒, 以及所需的量 (通常0.025 µg 到2.5 µg) 的效应质粒。加水, 使总容积达5µL。</li>
	<li>在发布的协议6之后, 准备1.6 µm 金载体 (参见材料目录)。</li>
	<li>对于每种治疗, 添加50µL 的悬浮载体到1.5 毫升管含有质粒 DNA。根据制造商的协议6, 将载体与质粒 DNA 一起涂上。</li>
	<li>在最后一步, 当质粒涂层的载体悬浮在48µL 100% 乙醇, 吸管 8 ul 悬浮载体到每五 macrocarriers 和允许他们空气干燥。</li>
</ol>4. Embryoless 大麦颗粒的微粒轰击
<ol><li>设置粒子轰击装置 (请参阅材料表)6。</li>
	<li>将 1550 psi 破裂盘放入固定帽, 并将其安装在仪器上。</li>
	<li>将含有所需质粒组合 (连同停止屏幕) 的 macrocarrier 放入载体发射组件中。将组件插入到轰击室的顶槽中。设置在标准距离 (6 毫米) 处的破裂盘护盖与 macrocarrier 盖盖之间的距离, 并将停止屏幕支持置于 (标准) 中间位置, 允许 macrocarrier 旅行距离为 11 mm (图 2A)。</li>
	<li>将 8 embryoless 颗粒排列在一个紧的径向模式下 (用较薄的一端指向内侧) 在滤纸圈上, 将其固定在一个 90 mm 培养皿的盖子上 (参见图 2B)。</li>
	<li>放置菜与五谷入炮击房间, 在距离从停止的屏幕 6 cm。</li>
	<li>将轰炸室疏散至95帕, 然后轰击样品。</li>
	<li>释放真空后, 将被轰击的谷物转移到一个标记为60毫米的培养皿中, 含有4毫升的饮用溶液, 含有氯霉素的1毫克/毫升。</li>
	<li>重复, 直到所有的轰炸已经完成。孵育 (在一个震动平台在 100 rpm) 在24°c 24 小时。</li>
</ol>5. 从轰击颗粒中提取可溶性蛋白质
<ol><li>使用镊子, 从60毫米的培养皿中取出8个被轰击的谷物, 并将它们涂抹在纸巾上晾干。将谷物分成两个标记为2.0 毫升的管子 (每管4粒), 每个含800个 ul 研磨缓冲器 (100 毫米 Na 磷酸盐 pH 值 7.2, 5 毫米, µg/毫升 leupeptin, 10 甘油) 和20% 毫米不锈钢珠。对所有被轰击的谷物重复这个过程。</li>
	<li>将 (最多 48) 2.0 毫升管放入珠均质机的两个机架上 (请参阅材料表)。将机架安装在均质机上。</li>
	<li>在30赫兹处摇动样品3分钟。</li>
	<li>从均质机卸下均质机架, 并将其放在冰上 (5 分钟) 以重新冷却样品。</li>
	<li>将机架重新安装在均质上-在相反的方向上。再次摇动样品在30赫兹3分钟。</li>
	<li>冷却在冰上的架子, 然后重复步骤5.3 到5.5。这将增加总共12分钟的震动。</li>
	<li>离心机的谷物提取物在 1.6万 x g 在4°c 10 分钟。</li>
	<li>
立即离心后, 醒酒清除上清液成第二组离心管。给每个管子一个快速的漩涡。把管子储存在冰上。</li>
	<li>转移上清液后, 从小球中回收钢珠, 以便清洗和再利用。</li>
</ol>6. 从谷物提取物中测定荧光素酶活性
<ol><li>要测定每个谷物提取管, 准备一个12毫米 x 75 毫米玻璃试管, 其中含有 200 ul 的荧光素酶检测混合物 (45 毫米三硫酸酯 pH 值 7.7, 15 毫米氯化镁2, 20 毫米, 1.5 毫米 EDTA, 1 毫米荧光素, 1 毫米 ATP)。</li>
	<li>将第一粒提取物的 100 ul 添加到第一检测管中。涡旋迅速搅拌良好。立即将管置于紫外线的管架中, 并关闭门。当测量完成后, 从紫外线中取下管子--让门打开, 以便放置下一管。</li>
	<li>重复此过程, 直到测量完所有样品。</li>
</ol>7. 谷物提取物中 GUS 活性的测定
<ol><li>添加 200 ul 的 GUS 化验缓冲器 (50 毫米 Na 磷酸盐 pH 7.2, 2.5 毫米 4-甲基伞形酮-葡糖苷酸, 10 毫米, 2 毫米 EDTA, 10 µg/毫升 leupeptin, 20% 甲醇, 0.02% 钠氮) 的每一个井的96个井板。</li>
	<li>将每个谷物提取物的 50 ul 放置到相应的井中。添加后与提示混合。用密封膜密封顶部。</li>
	<li>在37摄氏度在黑暗中孵化96井板20小时。</li>
	<li>离心机96井板在 4000 x g 在4°c 10 分钟并且安置它在冰。</li>
	<li>添加 250 ul 0.2 M Na2CO3到每个井的黑色96井板。</li>
	<li>将每离心 GUS 检测混合物的 6.25 ul 添加到相应的井中 (包含0.2 米 Na2CO3) 的黑色96井板。添加后与提示混合。包括水井与 6.25 ul 10 微米, 40 微米, 100 微米 4-甲基伞形酮作为标准。</li>
	<li>把黑盘子放进盘子荧光计, 读甲基伞形酮的荧光。以 360 nm 的激发波长和 460 nm 的发射波长进行读数。设置仪器的增益, 使含有 6.25 ul 的40微米 4-甲基伞形酮和 250 ul 的 0.2 MNa2CO 3 的油井读数为1000年荧光单位。</li>
</ol>8. 数据分析
<ol><li>在电子表格中输入荧光素酶和 GUS 化验的值。排除从考虑任何样本与荧光素酶读数低于2万相对发光单位 s-1, 因为这表明, 基因构造没有成功地传递到糊粉层细胞。</li>
	<li>对于每个成功轰击的谷物萃取物 (代表一组四 embryoless 谷物), 从原始 gus 和荧光素酶数据中计算规范化的 gus 表达式如下: 规范化 gus = [(gus 表达式 gus 空白) x (200万)]/(荧光素酶表达-荧光素酶空白)。 注意: 这将 GUS 活动的数量正常化为在一次轰炸中观察到的东西, 使荧光素酶读数为200万。</li>
	<li>报告一个特定的记者结构的相对水平在存在的效应构造或激素治疗正在测试通过比较的表现水平, 在没有效应或荷尔蒙。</li>
</ol>

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作者没有什么可透露的。

通过粒子轰击引入效应基因构造, 其次是记者构造的瞬态表达, 是解剖特定行为者在 GA/ABA 信号中的作用以及由此产生的激素调控基因的宝贵策略。表达. 然而, 在大麦糊粉层单元格中进行此类实验的现有 协议2, 3, 4, 5, 6 是非常劳力密集.每组被轰击的颗粒必须在砂浆和杵中手...

大麦糊粉层层是植物激素调控基因表达研究的一个成熟的模型系统1。特别是, 一些需要发芽或早期幼苗发育的基因由赤霉素 (GA) 激活, 而与应激反应相关的基因则由脱落酸 (ABA) 激活。ga 和 aba 信号通路是交织在一起的, 因为一些 ga 激活基因的表达被 ABA 抑制, 反之亦然1。
了解特定行为者在 GA/ABA 信号中的作用的一个有价值的策略是通过粒子轰击引入效应基因构造, 接着是记者构造的瞬态表达, 从而使结果影响下游基因表达的确定。使用报告基因, 如GUS (β-glucuronidase) 或荧光素酶允许对基因表达式的敏感和定量测量, 特别是在接收到效应器结构的细胞内。例如, 引入了一个对转录因子 TaABF15,6编码的效应器构造, 它建立了 ABA 诱导的基因, 如HVA1是由 TaABF1 诱导的, 而 GA 诱导的基因, 如Amy32b被压抑。粒子轰击作为一种实验策略已被多个实验室用来研究遗传/ABA 信号的不同方面。这项工作已导致识别启动子元素重要的激活两个 GA 诱导的2和 ABA 诱导的基因3, 并发现蛋白激酶4和转录因子5 , 调节这些基因的表达。
现有协议2,3,4,5,6为粒子轰击和随后的瞬态基因表达测量是相当劳动密集型的, 因为每套轰炸在砂浆和杵中, 几乎没有谷物是均匀的, 酶的测定是单独进行的。这篇手稿报告了一个改进的协议, 部分自动化和优化均匀化步骤和 GUS 化验, 以允许大量的吞吐量, 允许更多的治疗在同一实验中测试, 和/或包含更多的复制, 每种治疗获得更有统计学意义的结果。代表的结果显示为HVA1和Amy32b报告器构造的表达式, 由转录因子 TaABF1 以及 GA、ABA 和其他调控分子调控。

<table>
	<tbody>
		<tr>
			<td>GeneElute HP plasmid Maxiprep kit</td>
			<td>Sigma</td>
			<td>NA0310-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-vis spectrophotometer</td>
			<td>Nanodrop</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Himalaya barley grains</td>
			<td>/</td>
			<td>/</td>
			<td>A variety of hulless barley (store in the dark at 4&#176; C)</td>
		</tr>
		<tr>
			<td>sodium succinate</td>
			<td>Sigma</td>
			<td>S2378</td>
			<td>Reagent for Imbibing Solution</td>
		</tr>
		<tr>
			<td>calcium chloride (dihydrate)</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td>Reagent for Imbibing Solution</td>
		</tr>
		<tr>
			<td>Imbibing Solution</td>
			<td>home made</td>
			<td>/</td>
			<td>20 mM sodium succinate, 20 mM calcium chloride, pH 5.0. Sterilize by autoclaving before use.</td>
		</tr>
		<tr>
			<td>chloramphenicol</td>
			<td>Sigma</td>
			<td>C0378</td>
			<td>Prepare a 10 mg/mL stock solution in 70% ethanol.</td>
		</tr>
		<tr>
			<td>vermiculite</td>
			<td>Fisher</td>
			<td>NC0430369</td>
			<td>Used for vermiculite plates.</td>
		</tr>
		<tr>
			<td>filter paper circles (90 mm)</td>
			<td>Whatman</td>
			<td>1001 090</td>
			<td>Used for vermiculite and for pre-bombardment grain preparation</td>
		</tr>
		<tr>
			<td>Vermiculite Plates</td>
			<td>home made</td>
			<td>/</td>
			<td>Add 50 mL of vermiculite to a glass petri dish. Place a 90 mm paper circle on top of the vermiculite. Autoclave.</td>
		</tr>
		<tr>
			<td>forceps (fine pointed)</td>
			<td>Fisher</td>
			<td>13-812-42</td>
			<td>Used for removing seed coat from barley grains.</td>
		</tr>
		<tr>
			<td>forceps (ultra fine point)</td>
			<td>Fisher</td>
			<td>12-000-122</td>
			<td>Used for removing seed coat from barley grains.</td>
		</tr>
		<tr>
			<td>gold microcarriers (1.6 &#956;m)</td>
			<td>BioRad</td>
			<td>1652264</td>
			<td></td>
		</tr>
		<tr>
			<td>macrocarriers</td>
			<td>BioRad</td>
			<td>1652335</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chloride (dihydrate)</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td>Prepare a 2.5 M stock solution and store 1 mL aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>spermidine</td>
			<td>Sigma</td>
			<td>S0266</td>
			<td>Prepare a 100 mM stock solution and store 500 &#956;L aliquots at -20&#176; C (use within 2 months).</td>
		</tr>
		<tr>
			<td>rupture discs (1550 psi)</td>
			<td>BioRad</td>
			<td>1652331</td>
			<td></td>
		</tr>
		<tr>
			<td>stopping screens</td>
			<td>BioRad</td>
			<td>1652336</td>
			<td></td>
		</tr>
		<tr>
			<td>macrocarrier holders</td>
			<td>BioRad</td>
			<td>1652322</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolistic particle delivery system</td>
			<td>BioRad</td>
			<td>PDS-1000/He</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium phosphate monobasic monohydrate</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td>Reagent for 1M sodium phosphate pH 7.2</td>
		</tr>
		<tr>
			<td>sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S9763</td>
			<td>Reagent for 1M sodium phosphate pH 7.2</td>
		</tr>
		<tr>
			<td>1M sodium phosphate pH 7.2</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 6.9 g of sodium phosphate monobasic monohydrate with 7.1 g of sodium phosphate dibasic. Add water to 100 mL. Add NaOH to get pH 7.2.</td>
		</tr>
		<tr>
			<td>dithiothrietol (DTT)</td>
			<td>Sigma</td>
			<td>43819</td>
			<td>Dissolve in water to 1 M. Store at -20&#176; in 1 mL aliquots.</td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td>Dissolve in water to 10 mg/mL. Store at -20&#176; C.</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>Prepare a 50% solution in water.</td>
		</tr>
		<tr>
			<td>Grinding Buffer</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 10 mL of 1 M sodium phosphate pH 7.2, 500 &#956;L of 1 M DTT, 100 &#956;L of 10 mg/mL leupeptin, and 40 mL of 50% glycerol. Add water to 100 mL.</td>
		</tr>
		<tr>
			<td>stainlesss steel beads (5 mm)</td>
			<td>Qiagen</td>
			<td>69989</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL tubes</td>
			<td>Eppendorf</td>
			<td>22363352</td>
			<td>This specific model of tube is recommended for use with the homogenizer.</td>
		</tr>
		<tr>
			<td>bead homogenizer (TissueLyser)</td>
			<td>Qiagen</td>
			<td>85210</td>
			<td></td>
		</tr>
		<tr>
			<td>12mm x 75 mm glass test tubes</td>
			<td>Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>luciferin</td>
			<td>Goldbio</td>
			<td>LUCK-100</td>
			<td>Prepare a 25 mM stock solution and store 1 mL aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A7699</td>
			<td>Prepare a 100 mM stock solution and store 250 &#956;L aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>Reagent for 1M Tris sulfate pH 7.7.</td>
		</tr>
		<tr>
			<td>sulfuric acid</td>
			<td>Sigma</td>
			<td>258105</td>
			<td>Reagent for 1M Tris sulfate pH 7.7.</td>
		</tr>
		<tr>
			<td>1M Tris sulfate pH 7.7</td>
			<td>home made</td>
			<td>/</td>
			<td>Dissolve 12.1 g Tris base in 100 mL of water. Adjust pH to 7.7 with sulfuric acid.</td>
		</tr>
		<tr>
			<td>magnesium chloride</td>
			<td>Sigma</td>
			<td>M9397</td>
			<td>Dissolve in water to 2 M.</td>
		</tr>
		<tr>
			<td>Luciferase Assay Buffer (LAB)</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 3 mL of 1 M Tris sulfate pH 7.7, 500 &#956;L of 2 M magnesium chloride, 1 mL of 1 M DTT, and 200 &#956;L of 0.5 M EDTA. Add water to 50 mL.</td>
		</tr>
		<tr>
			<td>Luciferase Assay Mixture</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 15 mL of LAB, 800 &#956;L of 25 mM luciferin, 200 &#956;L of 100 mM ATP, and 4 mL of water. This makes enough assay mixture (20 mL) for 100 luciferase assays.</td>
		</tr>
		<tr>
			<td>luminometer (Sirius)</td>
			<td>Berthold</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>4-methylumbelliferyl-&#946;-D-glucuronide (MUG)</td>
			<td>Goldbio</td>
			<td>MUG1</td>
			<td>Dissolve in DMSO to 100 mM.</td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Sigma</td>
			<td>S8032</td>
			<td>Prepare a 2% stock solution in water and store 1 mL aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>96 well plates (standard)</td>
			<td>Fisher</td>
			<td>12565501</td>
			<td></td>
		</tr>
		<tr>
			<td>GUS assay buffer</td>
			<td>home made</td>
			<td>/</td>
			<td>Combine 2.5 mL of MUG, 5 mL of 1 M sodium phosphate pH 7.2, 400 &#956;L of 0.5 M EDTA, 1 mL of 1 M DTT, 100 &#956;L of 10 mg/ml leupeptin, 20 mL of methanol, and 1 mL of 2% sodium azide. Add water to 100 mL.</td>
		</tr>
		<tr>
			<td>TempPlate sealing film</td>
			<td>USA Scientific</td>
			<td>2921-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates (black)</td>
			<td>Costar</td>
			<td>3916</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium carbonate</td>
			<td>Sigma</td>
			<td>S7795</td>
			<td>Prepare a 200 mM solution in water.</td>
		</tr>
		<tr>
			<td>4-methylumbelliferone</td>
			<td>Sigma</td>
			<td>M1381</td>
			<td>Prepare a 100 &#956;M solution in water. Freeze 1 mL aliquots at -20&#176; C.</td>
		</tr>
		<tr>
			<td>microplate fluouresence reader</td>
			<td>Bio-Tek</td>
			<td>FLX-800</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring gene expression in bombarded barley aleurone layers with increased throughput

大麦籽粒糊粉层层是植物激素调控基因表达的重要模型体系。在糊粉层细胞中, 萌发或早期幼苗发育所需的基因由赤霉素 (GA) 激活, 而与应激反应相关的基因则由脱落酸 (ABA) 激活。通过粒子轰击引入糊粉层细胞的基因构造, 可以对 GA 和 ABA 信号的机制进行审问, 并利用酶法测定结果的瞬态表达。报告了一种改进的协议, 部分自动化和简化了谷物均匀化步骤和酶的测定, 使吞吐量大大超过现有方法。采用全自动组织均质器进行谷物样品的均匀化, 用96井板系统进行 GUS (β glucuronidase) 测定。使用该协议的代表性结果表明, 磷脂酶 D 活性可以发挥重要作用, 激活HVA1基因表达的 ABA, 通过转录因子 TaABF1。

本文所描述的技术可用于将任何试验基因结构引入大麦籽粒的糊粉层细胞中。然后可以方便地测量测试基因的表达水平 (图 2)。这里描述的高吞吐量协议大大提高了种子研磨和酶测定步骤的效率。该方法已用于评估转录因子 TaABF15、6的能力, 以激活 ABA 诱导的基因HVA17的表达。在这里...

Watch this Scientific Journal Video about Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput at JoVE.com

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

提出了一种改进的协议, 用于测量粒子轰击后大麦糊粉层细胞中的瞬时基因表达。采用全自动谷物研磨与96井板酶检测相结合, 为该过程提供了较高的吞吐量。

提高吞吐量的轰击大麦糊粉层层中基因表达的测定

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

measuring-gene-expression-bombarded-barley-aleurone-layers-with

Research

JoVE Journal

Genetics

6.4K 次观看.  Colby College. 提出了一种改进的协议, 用于测量粒子轰击后大麦糊粉层细胞中的瞬时基因表达。采用全自动谷物研磨与96井板酶检测相结合, 为该过程提供了较高的吞吐量。

视频: Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

科研

遗传学

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

通过两个互补细胞同步方案研究细胞周期调控的基因表达

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

无基因克隆的基因破坏的变形链球菌菌株的生成

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.

Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment.

一种基于昆虫蜕皮激素受体的奇异基因开关, 用于对转基因进行有鲁棒性、可逆性和可忽略的泄漏的刻意表达

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...