This work was supported by a grant from the Multiple Sclerosis Society of Canada. RWJ is the recipient of the Waugh Family MS Society of Canada Doctoral Studentship Award.

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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+purification+of+Escherichia+coli-expressed+human+thymosin-alpha1+using+affinity+chromatography+and+high-performance+liquid+chromatography.">Separation and purification of Escherichia coli-expressed human thymosin-alpha1 using affinity chromatography and high-performance liquid chromatography.</a> Protein Expr Purif. 77 (2), 140-145 (2011).</li><li>Tegel, H., Ottosson, J., Hober, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+the+protein+production+levels+in+Escherichia+coli+with+a+strong+promoter.">Enhancing the protein production levels in Escherichia coli with a strong promoter.</a> FEBS J. 278 (5), 729-739 (2011).</li><li>Akirav, E. M., Bergman, C. M., Hill, M., Ruddle, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+CD4(+)CD25(+)+T+cells+exacerbates+experimental+autoimmune+encephalomyelitis+induced+by+mouse,+but+not+rat,+antigens.">Depletion of CD4(+)CD25(+) T cells exacerbates experimental autoimmune encephalomyelitis induced by mouse, but not rat, antigens.</a> J Neurosci Res. 87 (15), 3511-3519 (2009).</li><li>Kapust, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+etch+virus+protease:+mechanism+of+autolysis+and+rational+design+of+stable+mutants+with+wild-type+catalytic+proficiency.">Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency.</a> Protein Eng. 14 (12), 993-1000 (2001).</li><li>Blommel, P. G., Fox, B. 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The authors declare no competing financial interests.

这里，我们已经描述了协议用于生产MOG 标记蛋白以及如何产生来自MOG 标签蛋白质纯MOG 1-125的。这个协议是基于两个标准His标签的蛋白质的纯化方法，以及用于一个较旧的基于MOG蛋白15的产生先前描述的方案。虽然没有在这里描述的，MOG 标签蛋白的主要用途是通过与蛋白质抗原免疫以诱导EAE。描述的EAE是如何在小鼠，这是与MOG 标签蛋白兼容诱导的协议，可以在参考3中找到。我们以前表明，免疫与MOG 标签衍生MOG 标签或MOG 1-125不仅诱导的CNS以更大的脊髓炎症和脱髓鞘自身免疫疾病的标准相比，MOG 35-55肽，也表明致病的IgH MOG B细胞塔吨认识MOG蛋白被激活，以产生响应于MOG 标签或MOG 1-125一个生发中心反应，但不MOG 35-55 5。因此，免疫接种MOG 标签确实诱导适当的抗MOG B细胞的反应。 MOG 标记蛋白是通过吸收到充电镍树脂（通过His标签）和洗脱纯化。因为在前面的步骤中生成的蛋白质的大量的，需要的吸收和洗脱的多轮收集大部分的蛋白质。我们已经发现，至少4个轮次都需要，如果使用的树脂的合适体积为50ml试管以分离出大多数蛋白质。如果需要的话，该协议可以扩展到使用更多或更大的管，更镍树脂来降低轮吸收的数量。可替代地，高效液相色谱（HPLC）与镍柱可有效地净化His-标记蛋白18 &lt;/ SUP&gt;而事实上我们发现，HPLC能够有效地净化MOG 标记蛋白（未发表意见）。通过HPLC不是许多标准免疫学实验室访问，这里列出的协议被设计为使用共同的实验室设备中进行。除了his-标签纯化后，MOG 标签蛋白确实含有一个S标签，如果优选的14，它与S-tag纯化协议兼容。 基于MOG（主要是人或大鼠）的重组蛋白之前已经8所述。这些是基于已自被取代由能够在大肠杆菌细菌产生较大量的蛋白质的强转录启动子驱动的系统较旧的表达系统。我们MOG 标签表达系统用于PET-32A（+）载体，它是显著比可用于内部生产MOG蛋白19的系统的效率高效T7启动子。然而，它那HOULD应当注意，这里所描述的MOG 标记蛋白是基于小鼠MOG。免疫与人MOG蛋白已经显示出诱导EAE中相比，使用鼠MOG 8引起的疾病具有不同的特征的小鼠。此外，鼠MOG可以比小鼠在某些情况下20小鼠MOG免疫原性更强。因此，对于一些基于小鼠实验目的MOG 标签可能不是很理想。我们是在产生几个不同版本的MOG 标签蛋白的比可能适合某些用途较好的方法中，并且这里所描述的纯化方案将所有的这些工作。在此期间，有可能的是，这里所描述的纯化方案可以针对其他MOG表达系统，或甚至其他蛋白质工作，只要它们掺入6×His标签吸收到镍树脂。然而，如果没有硫氧还蛋白标签，在较高浓度的MOG蛋白的溶解度可能是一个问题，当然就不可能戈<html> nerate纯MOG 1-125，因为这是唯一的MOG 标签系统。 测定蛋白质折叠之前通过透析MOG 标记蛋白浓度是该净化协议的成功是至关重要的。如果浓度过高，则蛋白质可以聚集和脱落的溶液代替折叠成单独的蛋白质。这可以在透析协议，为白色沉淀物在透析管的底部成形时可以看到。如果这发生，用6M胍再次溶解MOG 标签蛋白质和测量蛋白质浓度。稀释蛋白质到0.5毫克/毫升，然后再次启动透析如任何沉淀物应该形成于0.5毫克/毫升。对MOG 1-125，沉淀物可以预期，以形成作为所述蛋白质是高度不溶性的。我们已发现，这不会影响只要该蛋白前充分透析稀释MOG 1-125的折叠。 NT"&gt;对于TEV蛋白酶有效地切割MOG 标记蛋白进入纯MOG-125是使用TEV蛋白酶足够量的重要。TEV蛋白酶的旧版本是通过自我切割失活不过21更现代的版本患不溶性发出22因此重要的是要加入足够的TEV蛋白酶实现TEV蛋白酶失活前的MOG 标签蛋白质的足够的切割。由于市售TEV蛋白酶可能是昂贵的，我们建议生产和纯化TEV蛋白酶如在参照图22说明。当使用纯小鼠MOG 1-125蛋白质进行实验，要注意的是该蛋白是高度不溶性的，并且可以从溶液沉淀出来，因此必须进行广泛混合使用之前是重要的。 总之，在这里我们描述了一个简单协议，用于生产和提纯大量MOG 标记蛋白质。 Furtherm矿石，增加了一个TEV蛋白酶切割位点提供了MOG 标签蛋白提供了机会，如果需要的话，以产生纯MOG 1-125。 MOG 标签蛋白被抗自身免疫性的髓鞘B细胞和MOG诱导的标签采用EAE B细胞介导的病理5识别和结合。因此，MOG 标签蛋白克服的主要障碍限制了广泛使用的蛋白质抗原来诱导EAE。

MS是人类疾病的特点是慢性炎症和这被认为是由针对髓鞘自身免疫应答被驱动的中枢神经系统的神经变性。髓鞘和轴突随时间的流失导致认知和运动功能1逐渐下降。 "实验性自身免疫性脑脊髓炎"是用于针对CNS髓磷脂自身免疫性疾病的动物模型的总称。像人类的MS，EAE特征通常为CNS的免疫细胞浸润，并且在某些情况下，脱髓鞘2。然而，向其中任何给定的EAE模型类似于人的MS部分的程度取决于所使用的种或菌株和在底层的抗髓鞘自身免疫反应的复杂性。 抗髓鞘自身免疫可以通过实验诱导几种方式，但是目前使用的最常见的方法是将免疫小鼠用氨基酸模仿免疫的CD4的短肽<s了髓鞘蛋白&gt; + T细胞表位。这表示以诱导致病响应的最低要求。可能是最常见的这些是从髓鞘少突胶质糖蛋白（MOG 35-55）衍生的21氨基酸肽，它用于以诱导EAE在C57BL / 6小鼠3。然而，对于一些实验目的，希望或甚至必需的具有较大蛋白抗原和确实有一些优点这个过短肽免疫来免疫。第一，因为MHC限制的，短肽通常只有在菌株的一个非常有限的范围内有效，而代表任一整蛋白或特定域较大蛋白抗原可在不同的物种中正常处理以便呈现在多个近交小鼠品系或甚4。第二，较大的蛋白抗原能够诱导结合多种类型的淋巴细胞的抗原识别，更复杂的免疫反应，而不是limitin的15μg抗原识别到CD4 + T细胞。例如，通过其B细胞受体（BCR）的B细胞与整体，而不是处理的蛋白直接相互作用。我们和其他人已经表明由MOG 35-55免疫激活不承认MOG蛋白5 B细胞。由于B细胞最近证明在人MS 6发挥致病作用，即结合B细胞在自身免疫性病理学的EAE模型是越来越重要。 尽管使用较大的蛋白抗原以诱导EAE的优点，仍存在这样的蛋白质的一些可商购的来源。实际上，虽然短肽像MOG 35-55可以非常快速地并以相对低的成本来合成MOG蛋白的商业选择是有限的，成本基本上更购买。然而，存在可用于研究团体产生MOG胞外结构域的几个表达载体（MOG 1-125）本身。 HoweveR，所有我们在文献中已经确定了表达系统是基于至今已换成更高效表达系统7的旧技术。此外，大多数是基于大鼠或人MOG 8。用于在小鼠中自身免疫的一些调查，根据鼠标MOG自身抗原的抗原是优选的。最后，商业或作为表达载体，我们已经确定了所有基于MOG蛋白，是含有额外的氨基酸的MOG 1-125基的融合蛋白。这些措施包括净化，通常其他序列标签为好，这有很多的功能，我们无法辨认。 为了解决这些局限性，我们产生基于稠合到含有硫氧还蛋白打击MOG蛋白5的已知的不溶性的标记的小鼠MOG胞外结构域的新型融合蛋白。标签序列还包含用于净化和TEV蛋白酶CLE一个序列的6xHisavage站点，允许完全去除所有的标签序列的，如果需要的话。这是我们都知道的，生成纯MOG 1-125蛋白质的唯一方法。为了便于生产大量蛋白质的MOG 1-125序列用于细菌表达密码子优化和MOG 标签融合蛋白插入的pET-32表达系统。在这里，我们详细描述了该协议生产和净化MOG 标记蛋白，而纯MOG-125，使用现有的大多数实验室免疫学非专用设备。

<table border="0" cellpadding="0" cellspacing="0" width="1434">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">BL21 E.coli- pet32-MOGtag</td>
			<td style="width:139px;">Kerfoot lab</td>
			<td style="width:119px;"></td>
			<td style="width:803px;">These bacteria are required to make the MOGtag protein. Glycerol stocks of these bacteria are available upon request.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">LB broth miller</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">LBL407.1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">Ampicillin</td>
			<td style="width:139px;">bio basic</td>
			<td style="width:119px;">AB0028</td>
			<td>Reconsititute the powder into 50% ethanol/ 50% H2O at 100 mg/ml. Store at -20 &#176;C.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">IPTG</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">IPT002.5</td>
			<td>Reconsititute the powder into H2O at 1M and store at -20 &#176;C.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">Chicken-egg lysozyme</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">LYS702.10</td>
			<td>Reconstitute in H2O at 50 mg/ml and store at -80 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Triton-X100</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">T-8532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Phosphate buffered saline</td>
			<td style="width:139px;">life technologies</td>
			<td style="width:119px;">20012-027</td>
			<td>Commercial phosphate buffered saline is not required, any standard lab made phosphate buffered saline is sufficient.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium chloride</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SOD004.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris-HCl</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">TRS002.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Imidazole</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">IMD508.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Guanidine-HCl</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">G3272</td>
			<td>The quality must be greater than 98% purity.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">0.5M EDTA</td>
			<td style="width:139px;">bioshop</td>
			<td style="width:119px;">EDT111.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Nickel (II) sulfate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">NIC700.500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:375px;">His bind resin</td>
			<td style="width:139px;">EMD Millipore</td>
			<td style="width:119px;">69670-3</td>
			<td>Store in 20% ethanol 80% H2O at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Anhydrous ethanol</td>
			<td style="width:139px;">Commercial Alcohols</td>
			<td style="width:119px;">P016EAAN</td>
			<td>Dilute with water as needed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glacial acetic acid</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">ACE222.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sodium acetate trihydrate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SAA305.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">bovine serum albumin standard</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">500-0206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Bio-rad protein assay dye reagent concentrate</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:375px;">Ethylenediamine tetraacetic acid, disodium salt dihydrate</td>
			<td style="width:139px;">Fisher scientific</td>
			<td style="width:119px;">BP120-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Tris-base</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">TRS001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">7000 MW Snakeskin dialysis tubing</td>
			<td style="width:139px;">Thermoscientific</td>
			<td style="width:119px;">68700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">2-mercaptoethanol</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:119px;">M3148-25ml</td>
			<td>This reagent should not be handled outside of a fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">AcTEV protease</td>
			<td style="width:139px;">lifetechnologies</td>
			<td style="width:119px;">12575-015</td>
			<td>Producing your own TEV protease can be accomplished using (https://www.addgene.org/8827/) and purified as in reference 17</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Polyethyleneglycol 3350</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">PEG335.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">polyethyleneglycol 8000</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">PEG800.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Nunc MaxiSorp flat-bottom 96 well plate</td>
			<td style="width:139px;">ebioscience</td>
			<td style="width:119px;">44-2404-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Sonicator</td>
			<td style="width:139px;">Fisher scientific</td>
			<td style="width:119px;">FB-120-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Eon microplate spectrometer</td>
			<td style="width:139px;">Biotek</td>
			<td style="width:119px;">11-120-611</td>
			<td>This equipment uses the Gen5 data analysis software.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Gen5 data analysis software</td>
			<td style="width:139px;">BioTek</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">sodium dodecyl sulphate</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">SDS001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">bromophenol blue</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">BRO777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glycerol</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">GLY001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Protein desalting columns</td>
			<td style="width:139px;">Thermoscientific</td>
			<td style="width:119px;">89849</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Glycine</td>
			<td style="width:139px;">Bioshop</td>
			<td style="width:119px;">GLN001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">precast 12% polyacrylamide gel</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">456-1045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Rapid stain reagent</td>
			<td style="width:139px;">EMD Millipore</td>
			<td style="width:119px;">553215</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Gel dock EZ imager</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1708270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">White Light Sample Tray</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1708272&#160;</td>
			<td>Used along with gel dock EZ imager for coomassie blue stains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Protein ladder</td>
			<td style="width:139px;">bio-rad</td>
			<td style="width:119px;">1610375</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple and efficient production and purification of mouse myelin oligodendrocyte glycoprotein for experimental autoimmune encephalomyelitis studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

一旦纯化完成后，收集在步骤1.4，2.1，3.4的样品，并从步骤6.4最终产物应在蛋白凝胶（ 图3A）中运行。 MOG 标签应首先出现当T O / N样品中的31.86 kDa带，但不是T 0，并应在最终纯产物的唯一频带。为了测试MOG 标记蛋白是否已经正确折叠时，MOG 标签蛋白可用于通过FACS标记MOG蛋白特异性B细胞。通过用1标记小鼠淋巴细胞:MOG 标签 1000稀释用针对His标签和荧光标记的第三抗IgG1抗体的第二抗体一起MOG特异性B细胞可以识别（ 图3B）。可替代地，MOG 标签可直接缀合到荧光团以降低背景染色（从B细胞结合到二级和三级抗体产生）在参考5中所述，以确定特定MOG-B细胞。试图确定在野生型小鼠MOG结合B细胞时，因为这些细胞通常非常罕见5，这是必要的。的IgH MOG小鼠具有的重链敲入，当与适当的κ轻链配对，赋予特异性MOG。由于MOG 标签的结合是室内运动场MOG B细胞之间增强，这证实了这个协议产生正确折叠的抗原。重要的是，的IgH MOG B细胞向自身免疫病理学中MOG定向的EAE 12的模型中，确认了MOG 标签诱导T和B细胞自身免疫。 开始透析如第5说明再折叠蛋白质之前，有必要使用Bradford测定法来测量蛋白质浓度，如表1第4所述Bradford测定法的代表性结果示</s仲&gt;并总结在图5中。 纯MOG 1-125的产生通过加入TEV蛋白酶的对MOG 标签蛋白最终导致了MOG 标记蛋白的裂解成MOG 1-125和标签序列， 如图6完成。随后镍树脂纯化除去MOG 标签 ，标签序列，和TEV蛋白酶杂质最终导致纯MOG 1-125， 如图6。 <img alt="图1" src="/files/ftp_upload/54727/54727fig1.jpg" /> 图1:MOG 标记蛋白 （经许可这里重复的从Dang 等5。）。直链结构，以及氨基酸和MOG 标签融合蛋白的DNA序列。小鼠MOG的胞外域的DNA序列（MOG1-125，在蓝色下序列）用于在细菌中表达（黑色密码子优化）。该序列的合成和插入载体来创建的N末端融合到包含硫氧还蛋白和S-标签以抵消MOG蛋白13,14的已知的不溶性的标记，以及6倍他对纯化15标记。一个TEV蛋白酶裂解位点从标签序列分离MOG 1-125。使用替代共识TEV裂解位点16的结果清除所有非MOG氨基酸TEV介导谷氨酰胺-164和甘氨酸-165之间的分裂。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54727/54727fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54727/54727fig2.jpg" /> 图2:以生产纯MOG 标记蛋白质所需的步骤概述要。生成MOG 标签蛋白质，表达MOG 标签蛋白的细菌生长到高密度，然后诱导表达使用IPTG MOG 代码作为第1所列的过夜培养后，将细菌裂解，并通过一系列造粒的步骤包含蛋白质级分包涵体，其中包含MOG 标签 ，提取为第2节中MOG 标签上市，然后从粗蛋白组分纯化通过吸收的四个周期到充电镍树脂和MOG 标签蛋白洗脱在第3节的部分上市的汇集洗脱液然后采取Bradford测定法来确定MOG 标记蛋白质的产率和洗出液的其余部分被透析入乙酸盐缓冲液在数天的过程中如在第4列和5.最后，将蛋白质浓缩使用PEG 3350和PEG 8000至5毫克的最终浓度/ ml的根据MOG 标签的在B所确定的产量莱福法在第6节中列出的整个过程需要至少10天，开始每天在括号中的每个步骤。然而，另一种停止和启动点在协议中列出，并且步骤可以遍布的如果需要的时间量更大。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54727/54727fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54727/54727fig3.jpg" /> 图 3: 在MOG 标签蛋白质和其活性的评估的纯化 （A）中所示的是来自不同点收集跨越蛋白质纯化过程，并在SDS-PAGE凝胶上运行的蛋白质样品。 T 0的=前蛋白诱导（步骤1.4收集）BL21细菌，TØ蛋白表达/ N = BL21菌诱导后（收集在步骤2.1），原油MOG 标签 =溶解的MOG 标记蛋白之前，蛋白质纯化（步骤3.4收集），纯MOG 标签 = MOG 标记蛋白质纯化（步骤6.4收集后）。 （B）从野生型C57BL / 6小鼠或表达特异于MOG蛋白7,17免疫球蛋白重链的IgH MOG小鼠MOG 标签蛋白质从淋巴结CD19 POS CD4 负幼稚B细胞的结合用流式细胞术评估。 MOG 标签特异性的B细胞被染色的淋巴结细胞用MOG 标签后跟一个第二抗his标签抗体和荧光叔抗IgG1抗体识别。从C57BL / 6或IgH基因小鼠MOG细胞的染色与MOG的IgH细胞的MOG 标签荧光减一（FMO）控制染色一同显示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54727/54727fig3large.jpg" target="_blank">请Ç</a>舔此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54727/54727fig4.jpg" /> 图4:步骤概述，生成MOG 1-125 从MOG 标签 的蛋白质。如在该协议的步骤3.5中所述收集MOG 标签洗脱液后，MOG 标签蛋白质透析到TEV蛋白酶裂解缓冲液中。一旦透析完成后，TEV蛋白酶加入到导致MOG 标签的裂解成MOG 1-125蛋白质的MOG 标记溶液。然后将裂解的溶液的体积减少，并透析到前蛋白质纯化缓冲液B。从裂解溶液中的杂质，通过四个连续几轮吸收到充电镍树脂和最终导致纯MOG的溶液中的杂质溶出萃取&lt;<html>子&gt; 1-125。在MOG 1-125蛋白的浓度通过Bradford测定法确定，并且该蛋白被折叠在数天通过透析的过程中。一旦透析完成后，MOG-125蛋白浓缩至2.24毫克/毫升使用PEG 3350和PEG 8000。该协议部分中，详细讨论7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54727/54727fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54727/54727fig5.jpg" /> 图5: 用于确定MOG 标记的浓度的Bradford测定标准曲线的实施例 蛋白。从表1中所采取的BSA标准读数作图，得到的线性回归公式计算基于在595nm处的光密度的MOG 标记浓度。F ="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54727/54727fig5large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图6" src="/files/ftp_upload/54727/54727fig6.jpg" /> 图6:MOG 标记 蛋白 的TEV切割 和MOG 1-125 的后续纯化所示的是蛋白质样品上MOG 1-125的SDS-PAGE凝胶示范纯化运行纯MOG 标记 =到TEV切割之前（收集MOG 标记蛋白。在步骤6.4），MOG 标签瓦特/ TEV =蛋白组分被后TEV蛋白酶MOG 标签培养72小时收集（收集在该MOG 1中保持绑定到镍树脂步7.5）=洗脱蛋白组分125纯化方案（步骤7.9收集），纯MOG 1-125 = MOG净化（收集步骤7.12）之后1-125蛋白。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54727/54727fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> BSA（毫克/毫升） </td><td> 0.4 </td><td> 0.35 </td><td> 0.3 </td><td> 0.25 </td><td> 0.2 </td><td> 0.15 </td><td> 0.1 </td><td> 0.05 </td><td> 0 </td></tr><tr><td></td><td> 1.079 </td><td> 0.998 </td><td> 0.948 </td><td> 0.853 </td><td> 0.769 </td><td> 0.699 </td><td> 0.583 </td><td> 0.493 </td><td> 0.373 </td></tr><tr><td></td><td> 1.071 </td><td> 1.014 </td><td> 0.95 </td><td> 0.854 </td><td> 0.777 </td><td> 0.681 </td><td> 0.579 </td><td> 0.484 </td><td> 0.375 </td></tr><tr><td></td><td> 1.069 </td><td> 1.017 </td><td> 0.944 </td><td> 0.848 </td><td> 0.781 </td><tD&gt; 0.687 </td><td> 0.592 </td><td> 0.494 </td><td> 0.374 </td></tr><tr><td> MOG 标签稀释</td><td> 1 </td><td> 1/5 </td><td> 1/25 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> 1.327 </td><td> 1.013 </td><td> 0.493 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> 1.332 </td><td> 1.063 </td><td> 0.491 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> 1.367 </td><td> 1.088 </td><td> 0.488 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表1:从Bradford测定代表值确定MOG 标记 蛋白 的浓度的BSA DILU。在已知浓度的系统蒸发散被用于确定在图5所示的标准曲线MOG 标签蛋白质交纯化的稀释液被用于确定最终MOG 标签蛋白质浓度。行包含在595nm测量的光密度和每行代表每一指明浓度共3个重复中的一个复制。

Watch this Scientific Journal Video about Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies at JoVE.co

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

简单而高效的生产和鼠标髓鞘少突胶质糖蛋白的纯化的实验性自身免疫性脑脊髓炎研究

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

simple-efficient-production-purification-mouse-myelin-oligodendrocyte

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.9K 次观看.  Western University. We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

视频: Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

鼠标垂体免疫原的制备诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

科研

免疫与感染

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJL小鼠诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

在实验性自身免疫性脑脊髓炎（EAE）抗性品系小鼠继转移和抗原挑战克服反应迟钝

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

髓鞘少突胶质细胞糖蛋白（MOG<sub&gt; 35-55</sub&gt;）诱导实验性自身免疫性脑脊髓炎（EAE）的C57BL / 6小鼠

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

实验性自身免疫性葡萄膜炎:眼内炎症小鼠模型

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

小鼠不变自然杀伤性T细胞的纯化和扩增用于体外和体内研究

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...