这项工作部分得到了中国自然科学基金资助（授权号31471703，合资和律师事务所A0201300537和31671854）以及100名外籍人才计划（授予合资公司JSB2014012）。

涉及动物受试者的程序已经根据"国家实验动物福利准则"（中国科学技术部，2006年），南京农业大学实验动物中心伦理委员会批准，动物饲养在SPF设施（Permission ID:SYXK-J-2011-0037）。 Cmah敲出鼠标模型<ol><li>使用扬州大学比较医学中心野生型C57Bl / 6小鼠（中国）。 注意:基于商业上使用CRISPR / Cas9 20战略来自先前CMAH敲除研究17，第 19和获得所提供的信息通过从所述CMAH基因，这也是外显子6中除去92个碱基对产生CMAH敲除小鼠在人CMAH基因中被删除19 。正F 0 &lt;/子&gt; -mice（2个个体）与野生型小鼠杂交以获得杂合f 1的-mice。杂交F 1 -小鼠（5个个体）杂交后，可以成功获得纯合Cmah敲除F 2 -小鼠（3个个体）。 </li><li> 使用商业DNA纯化试剂盒，根据Zangala 21所示的方法，使用基因组DNA进行小鼠的基因分型。 <ol><li>通过使用商业DNA聚合酶和引物对5&#39;gaaagggctcggctctgtatgaa3&#39;和5&#39;tttaaaatgtcccgggtgagaagc3&#39;扩增相应的Cmah基因DNA片段。使用34个PCR循环进行基因扩增，94个循环由94℃变性40秒，63℃退火40秒，72℃延伸1分钟。 </li><li>在1％琼脂糖凝胶上显示PCR产物。应该观察野生型个体的单条带（0.5kb），双带（0.4和0.5kb）杂合敲除个体，以及纯合敲除个体的单条带（0.4kb）。 </li></ol></li></ol>样品收集<ol><li>使用Willingham 等人描述的方案收集小鼠牛奶22 。 </li><li>使用Gonçalves 等人描述的方案收集小鼠肝组织23 ，并将样品储存在-80°C。 </li></ol> 3.从牛奶中分离唾液酸<ol><li>通过添加12毫升冰醋酸的制备100毫升2M的乙酸溶液加入到88毫升蒸馏水H 2 O的</li><li>将50μL牛奶转移到1.5 mL离心管中，加入1.2 mL制备的乙酸水溶液。 </li><li>在80℃下将悬浮液孵育4小时，并以14,000xg离心10分钟。 </li><li>将顶部1000μL的上清液转移到新鲜的1.5mL离心管中，在室温下通过离心蒸发除去溶剂以完成干燥（取决于所附的真空泵，这将需要4-20小时）。重新溶解在蒸馏水H 2 O的500μL的所述样品</li><li> 制备微量阴离子交换柱。该步骤将特别富集阴离子化合物并从乳和肝样品中除去阳离子和中性化合物，因此增加了荧光OPD标记剂对唾液酸衍生化的选择性。 <ol><li>将每个样品200mg的阴离子交换树脂（Dowex 1X8）转移到具有连接的旋塞的空的3mL柱管中。 </li><li>加入2mL在步骤3.1中制备的乙酸水溶液以用乙酸根离子代替树脂中的氯离子。一旦溶剂到达树脂顶部就关闭活塞。 </li><li>轻轻加入2毫升蒸馏水2 O的，打开活塞，只要大部分溶剂达到的停止流动树脂的顶部。确保树脂总是被溶剂覆盖。 </li><li>两次用2mL蒸馏水2 O的洗涤树脂柱以除去乙酸过量。 </li></ol></li><li>从步骤3.4转移所得的500μL样品溶剂。到树脂柱上并丢弃流通。轻轻用2mL蒸馏水H 2 O洗脱使用50 mM乙酸铵溶液的1毫升的树脂样品阴离子（386毫克乙酸铵溶解在100毫升蒸馏水2 O的）的洗涤树脂到1.5mL离心管。通过在室温下离心蒸发除去溶剂至干。 注意:干燥样品可以在4-6°C下储存长达12个月。 </li></ol> 4.从肝组织中分离唾液酸<ol><li>轻轻地解冻20 - 50毫克小鼠肝脏，并将其转移到Dounce组织研磨机（1 mL或2 mL体积）中。加入1.2mL乙酸水溶液在步骤3.1中制备并通过温和剪切将组织匀浆10秒。将所得悬浮液转移到1.5 mL离心管中。 </li><li>按照步骤3.3。至3.6。 注意:干燥样品可以在4-6°C下储存长达12个月。 </li></ol> 5.制备混合唾液酸标准品<ol><li>将5-8mg N-乙酰神经氨酸在分析天平称重至1.5mL离心管中。注意准确的重量，并添加蒸馏水2 O的164μL每毫克（ 即 ，如果的Neu5Ac的重量为7.2毫克再加入7.2×164 = 1，蒸馏水2 O的181微升）。这产生20mM Neu5Ac储备溶液，其可以在-20℃下储存多达12个月。 </li><li>以适量的价格获得较小量的N-羟乙酰神经氨酸，例如1mg等分试样。 2 O直接添加154μL蒸馏水到化合物中，以获得〜20mM的Neu5Gc的储备溶液。转移溶液加入1.5 mL离心管中。该溶液可以在-20°C储存长达12个月。 </li><li>将来自步骤5.1和5.2的每种储备溶液中的5μL合并到新鲜的1.5mL离心管中，并通过在室温下离心蒸发除去溶剂至干。 注意:混合唾液酸标准品可以在4 - 6°C下储存长达12个月。 </li></ol>唾液酸荧光衍生化<ol><li>制备10mL的OPD溶液，由100毫克邻苯二胺和在10mL的208毫克亚硫酸氢钠的蒸馏水H 2 O. </li><li>向来自小鼠乳（步骤3），小鼠肝脏（步骤4）或混合唾液酸标准品（步骤5）的唾液酸样品中加入20μLOPD溶液。涡流在80℃下在黑暗中大力进行30秒，并培育样品4小时（ 即包裹在铝箔微管）。 </li><li>让样品冷却5分钟，加入80μ;蒸馏水2 O的L和在14,000×g离心试管1分钟。 </li><li>将80μL的上清液转移到300μL高回收率的HPLC样品瓶中。衍生的唾液酸样品可以在4-6℃下储存长达一周。 </li></ol> 7.唾液酸衍生物的HPLC分析<ol><li>使用连接到在线荧光检测器的标准HPLC系统分析样品。 </li><li>使用反相C18柱，标准尺寸为250 mm，直径为4.6 mm，用于分析。 </li><li> 通过用800mL的LCMS级水（LCMS-液相色谱质谱法）稀释200mL储备溶液来制备溶剂A.储备液本身可以制备如下: <ol><li>加入46克甲酸至800毫升的LCMS级H 2 O. </li><li>通过滴加氢氧化铵溶液（puriss。pa）将pH调节至4.5。 </li><li>将溶剂转移至量筒，并用LCMS级H 2 O填充至1000 mL。该储液可在4 - 6°C下储存3个月。 </li></ol></li><li>对于溶剂B，使用LCMS级乙腈。 </li><li> 以1 mL / min的流速与以下梯度洗脱分离唾液酸衍生物: <ol><li>首先加入混合溶剂A的10％溶剂B. </li><li>从0分钟到15分钟，用线性梯度逐渐增加溶剂B的比例至60％。 </li><li>从15分钟到16分钟，以60％至90％的线性梯度迅速增加溶剂B的比例。这启动了HPLC柱的洗涤。 </li><li>为了进一步洗涤HPLC柱，将溶剂B的水平在16分钟和18分钟之间保持在90％，然后在18分钟和19分钟之间再次将溶剂B的水平再次降低到10％。 </li><li>重新平衡HPLC色谱柱至10％B的起始条件为19至24分钟。 </li></ol></li><li>在将50μL样品注入HPLC系统。 </li><li>使用荧光检测器的激发/发射波长为373/448nm监测洗脱液，衍生的唾液酸可以在9分钟的近似保留时间（对于Neu5Gc-OPD）和10分钟（对于Neu5Ac-OPD）来说是预期的。 </li><li>从Neu5Ac（A Neu5Ac ）和Neu5Gc（A Neu5Gc ）的荧光峰面积计算Neu5Gc（F Neu5Gc ）的相对量如下:F Neu5Gc [％] = 100×A Neu5Gc /（A Neu5Ac + A Neu5Gc ）。在来自纯合Cmah敲除小鼠F Neu5Gc的牛奶和肝脏样品的情况下，杂合子和野生型小鼠的F Neu5Gc的值可以根据小鼠年龄和组织类型（2％和&gt; 90％），预期误差幅度为±12％。 </li></ol>

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作者没有什么可以披露的。

本文提出的方案允许通过分析和定量牛奶和肝脏样品的Neu5Gc的相对量来对纯合Cmah敲除小鼠进行表型评估。使用具有荧光检测的标准HPLC装置进行分析。该方法最关键的步骤是制备阴离子交换柱并进行阴离子交换层析;妥善安置树脂并收集正确的洗涤和洗脱部分需要一些练习。 或者，本文使用的衍生化试剂OPD可以容易地用更昂贵的衍生剂DMB（1,2-二氨基-4,5-亚甲基二氧基苯?...

N-乙酰神经氨酸（Neu5Ac）和N-羟乙酰神经氨酸（Neu5Gc）是大多数哺乳动物中最常见的唾液酸1 。虽然能够内源性合成的Neu5Ac，人类不能产生Neu5Gc的由于对CMAH编码用于CMP-Neu5Ac的羟化酶2,3该基因的初级外显子缺失。然而，动物类食品产品可以Neu5Gc的4，5，6的饮食来源，导致产生抗Neu5Gc抗体，并因此触发朝向的Neu5Gc 7的免疫应答。 Neu5Gc的这一膳食效果被怀疑向慢性炎症和各种其它疾病8，9，10。为了全面了解Neu5Gc在h中的作用umans，一种用于系统研究食源性唾液酸影响的动物模型是非常需要的。虽然基于聚合酶链反应（PCR）分析敲除小鼠的方案已经很好地建立，并且是基因型评估的便捷方式，但代谢水平上表型的功能分析需要更具体的分析方法。可以通过分离和分析肝脏或牛奶样品中唾液酸的组成来评估Cmah敲除小鼠模型的表型。以前已经报道了几种用于检测动物组织中唾液酸的方法:唾液酸与间苯二酚11或硫代巴比妥酸12反应导致形成发色产物，并且可以使用基于平板印刷机的设置进行简单的分析，但只有总唾液酸可以测定酸含量。或者，也使用气相色谱法描述唾液酸的分析13 ，MALDI-ToF质谱法14或电流法15 。然而，最常用的唾液酸分析方法是基于水解释放，随后荧光衍生化和随后的高效液相色谱16，17，18。

<table border="0" cellpadding="0" cellspacing="0" width="684">
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			<td height="20" style="height:20px;width:147px;">Chemicals:</td>
			<td style="width:144px;"></td>
			<td style="width:143px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-acetylneuraminic acid</td>
			<td>Sigma</td>
			<td>A0812</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-glycolylneuraminic acid</td>
			<td>Sigma</td>
			<td align="right">50644</td>
			<td>1 mg aliquot should be sufficient</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">o-Phenylenediamine</td>
			<td>Sigma</td>
			<td align="right">694975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydrogen sulfite</td>
			<td>J&#38;K Scientific Ltd</td>
			<td align="right">75234</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tools/Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3 mL SPE tubes</td>
			<td>Supelco</td>
			<td>Sigma 57024</td>
			<td>empty solid phase extraction columns</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luer stopcock</td>
			<td>Sigma</td>
			<td>S7396</td>
			<td>to stop the flow of the SPE tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dowex 1X8</td>
			<td>Dow Chemicals</td>
			<td>Sigma 44340</td>
			<td>200-400 mesh</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dounce tissue grinder</td>
			<td>Sigma</td>
			<td>D8938</td>
			<td>tight fit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Analysis:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High-recovery HPLC vial</td>
			<td>Agilent Technologies</td>
			<td>#5188-2788</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC System</td>
			<td>Shimadzu</td>
			<td>Nexera</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Detector for HPLC</td>
			<td>Shimadzu</td>
			<td>RF-20Axs</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Column</td>
			<td>Phenomenex</td>
			<td>Hyperclone ODS</td>
			<td>250x4.6 mm</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LCMS-grade H2O</td>
			<td>Merck Millipore</td>
			<td>#WX00011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Acetonitrile</td>
			<td>Merck Millipore</td>
			<td>#100029</td>
			<td>Hypergrade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium hydroxide solution</td>
			<td>Fluka</td>
			<td>#44273</td>
			<td>puriss. P.a.</td>
		</tr>
	</tbody>
</table>

determination of sialic acids in liver and milk samples of wild-type and cmah knock-out mice.

CMAH（胞苷一磷酸-N-乙酰神经氨酸羟化酶）负责哺乳动物中胞苷一磷酸-N-乙酰神经氨酸的氧化。然而，由于CMAH基因的主要外显子缺失，人类不能将胞苷一磷酸-N-乙酰神经氨酸氧化为胞苷一磷酸-N-羟乙酰神经氨酸。为了更清楚地了解CMAH活动缺乏的影响和影响，小鼠中的Cmah敲除模型对基础和应用研究非常感兴趣。本文详细描述了确定该小鼠模型的表型的分析方法，并且基于在野生型和Cmah敲除小鼠的肝脏和牛奶中检测到N-乙酰神经氨酸和N-羟乙酰神经氨酸。内源性唾液酸被邻苯二胺释放并衍生，产生荧光衍生物，随后可用HPLC分析。所呈现的协议可以是也适用于各种其他来源的牛奶和组织样品的分析，可用于研究N-羟乙酰神经氨酸的营养和健康影响。

所描述的分析方法的示意图如图1所示，包括从野生型和Cmah敲除突变小鼠的牛奶和肝脏样品中分离唾液酸，以及这些组分的荧光衍生化和HPLC分析。 图2和图3显示了来自同源和杂合敲除Cmah小鼠（ - / - 和+/-）和野生型小鼠的牛奶和肝脏样品的衍生唾液酸的代表性HPLC色谱图</strong...

Watch this Scientific Journal Video about Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice. at JoVE.com

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

我们描述了一种基于HPLC的方法，用于测定小鼠肝脏和牛奶中的N-乙酰神经氨酸和N-羟乙酰神经氨酸。

测定野生型肝脏和牛奶样品中的唾液酸<em&gt; CMAH</em&gt;敲出小鼠

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

determination-sialic-acids-liver-milk-samples-wild-type-cmah-knock

Research

JoVE Journal

Biology

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

9.7K 次观看.  Nanjing Agricultural University. 我们描述了一种基于HPLC的方法，用于测定小鼠肝脏和牛奶中的N-乙酰神经氨酸和N-羟乙酰神经氨酸。 

视频: Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

定量组织样本中的γH2AX灶

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

科研

生物学

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

使用内源性野生型BRCA1基因的细胞表达，确定同源重组的BRCA1基因突变的影响

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

使用微量的生物规格的纳米分光光度计核酸和蛋白的浓度测定

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

在体内肝细胞的分离纯化的肝细胞内吞其次是肝脏灌注

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

一个基于PCR的基因分型方法来区分野生型和观赏品种白茅</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

牛奶收集方法小鼠和里夫斯&#39;麂鹿

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

奶源在大鼠毛细管管和乳脂肪含量Creamatocrit估计

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...