作者感谢妮可 McRorie 的帮助与实验。这项工作得到了阿尔伯塔大学医学院和牙科学院的支持, Slipchuk SMA 研究基金会研究基金, 加拿大卫生研究院 (卫生研究院), 加勒特的朋友卡明研究基金会, Toupin 神经学科研经费、加拿大肌肉萎缩症、加拿大创新基金会、艾伯塔省企业和高级教育以及妇女儿童健康研究所 (WCHRI)。

1. 细胞培养
<ol><li>培养 SMA 患者成纤维细胞生长培养基 (Dulbecco 的改良鹰中等 1:1 F-12 (火腿) 与10% 胎牛血清 (血清) 和0.5% 青霉素/链霉素) 在 CO2孵化器在37°c。</li>
	<li>使用自动细胞计数器和稀释细胞到生长培养基中的 1 x 105细胞/mL, 然后在适当的板块上播种, 确定细胞浓度。使用12井板 (每井1毫升) 用于 rt-pcr 或 qPCR。</li>
	<li>在 CO2孵化器中, 在37摄氏度孵育24小时的板材。</li>
</ol>2. 低噪声/DNA mixmer 转染
<ol><li>确保细胞融合约 65-80%, 以获得最佳转染效率。</li>
	<li>在冰上慢慢解冻10微米的低噪声或 DNA mixmers。</li>
	<li>在一个1.5 毫升的管, 准备一个 20x mixmer 解决方案通过添加 mixmers 到血清缺乏的媒体, 使最终体积达到 50 ul。</li>
	<li>将 mixmer 溶液与含有3% 转染试剂 (1:1 比值) 的血清缺乏介质的 50 ul 混合在一起。</li>
	<li>室温孵育10分钟。</li>
	<li>添加 900 ul 血清缺乏培养基, 每管5% 只。</li>
	<li>从盘子中吸取旧的介质, 并用1毫升的低噪声/DNA mixmer 溶液取代含有转染试剂。</li>
	<li>在 CO2孵化器中孵化 24-48 小时37摄氏度的细胞。</li>
</ol>3. RNA 提取
<ol><li>去除培养基并加入1毫升的硫氰酸胍-苯酚-氯仿试剂到细胞中。用胍硫氰酸盐-苯酚-氯仿多次冲洗井底, 并将其收集成1.5 毫升管。</li>
	<li>漩涡在高速三十年代和存放在-80 °c 1 小时或隔夜。</li>
	<li>在室温和涡旋井中解冻样品。</li>
	<li>加200µL 氯仿, 用力摇动十五年代, 室温孵育3分钟。</li>
	<li>离心机在 1.2万 x g 15 分钟在4摄氏度。</li>
	<li>把最清澈的水相收集到新的管子里。确保避免收集在上下相之间形成的任何白色相间的界面。</li>
	<li>添加500µL 异丙醇和1µL 8 µg/µL 糖原 (RNA 级)。漩涡十年代和在室温孵育10分钟或在-20 °c 过夜。</li>
	<li>离心机在 1.2万 x g 10 分钟在4°c 和去除所有上清。在管子的底部会形成一个白色的小球。</li>
	<li>添加1毫升冷75% 乙醇和离心机在 7500 x g 5 分钟在4°c。</li>
	<li>除去所有的乙醇, 在室温下将颗粒干燥, 直到管子里没有乙醇。</li>
	<li>在30µL DNase/RNase 游离水中溶解颗粒, 在60摄氏度时孵育10分钟。</li>
	<li>使用分光光度计测量 rna 浓度 (260 nm), 并将 rna 浓度调整为50µL。</li>
</ol>4. 一步 rt-pcr 测量SMN2的外显子夹杂率
<ol><li>混合12.5 µL 的 2x rt-pcr 反应缓冲器, 1.0 µL 的一步 rt-pcr 酶混合, 0.5 µL 每个底漆 (SMN2向前: 5 '-CTGCCTCCATTTCCTTCTG-3 ', SMN2反向: 5 '-TGGTGTCATTTAGTGCTGCTC-3 ', GAPDH向前:5 '-TCCCTGAGCTGAACGGGAAG-3 ', GAPDH反转: 5 '-GGAGGAGTTTGGTCGCTGT-3 ', 2 µL 总 RNA (50 ng/µL), 8.5 µL RNase/DNase 无蒸馏水。</li>
	<li>在50°c 合成了15分钟的 cDNA 后, 开始 PCR 反应。放大SMN2外显子6-8 与 30 PCR 周期 (94 °c 为十五年代, 60 °c 为三十年代, 68 °c 为二十五年代)。放大 GAPDH 与 20 PCR 周期 (94 °c 为十五年代, 60 °c 为三十年代, 68 °c 为二十年代)。</li>
	<li>将 5 ul 6x 负载染料添加到 PCR 产品中, 并将混合物的5µL 装入2% 琼脂糖凝胶中, 并在 100 v 上运行40分钟。</li>
	<li>用 ImageJ 软件对凝胶进行图像分析。</li>
	<li>测量全长带和Δ7带的强度。根据 (全长)/(全长 + Δ7 SMN2) 的强度值计算外显子7包含的速率。</li>
</ol>5. 定量 PCR (qPCR) 测量SMN2的外显子夹杂率
<ol><li>对于 cDNA 合成, 混合1µL 寡核苷酸 dT, 1 µL 10 毫米 dNTPs, 11 µL 总 RNA (50 ng/µL)。孵育65°c 5 分钟。</li>
	<li>将样品放在冰上, 并添加4µL 5x 缓冲器, 1 µL 0.1 米, 1 µL RNase 抑制剂, 1 µL 逆转录酶对每个样品。</li>
	<li>孵育在50°c 60 分钟, 加热高达80°c 10 分钟, 以停止反应。该 cDNA 可以储存在-20 摄氏度。</li>
	<li>用无 RNase 水稀释基因 (1:5 稀释)。混合10µL 2x SYBR 绿色 qPCR 酶混合, 0.8 µL 10 µM 每个底漆 (全长SMN2向前: 5′-GCTATCATACTGGCTATTATATGGGTTTT-3′, 全长SMN2反转: 5′-CTCTATGCCAGCATTTCTCCTTAAT-3′, Δ7 SMN2前进: 5′-TCTGGACCACCAATAATTCCCC-3, Δ7 SMN2反向: 5′ ATGCCAGCATTTCCATATAATAGCC 3′, GAPDH向前: 5′ GCAAATTCCATGGCACCGT-3′, GAPDH反转: 5′-AGGGATCTCGCTCCTGGAA 3′), 3 µL 稀释 cDNA, 5.4 µL 无 RNase 水。</li>
	<li>开始 qPCR 反应。使用条件:95 °c 为三十年代, 然后40周期重复在95°c 为 5 s 和60°c 二十年代。</li>
	<li>计算全长SMN2到GAPDH或Δ7 SMN2的相对表达式, 并使用ΔΔCt 算法正常化到未处理的单元格。</li>
</ol>6. 西方印迹
<ol><li>从细胞中取出转染培养基, 再用磷酸盐缓冲盐水 (PBS) 冲洗一次。</li>
	<li>用蛋白酶抑制剂添加100µL 裂解缓冲液。</li>
	<li>用无菌的细胞刮刀从每个井的底部分离出细胞, 并收集成1.5 毫升的管子。在冰上孵化样品30分钟。</li>
	<li>通过21克针通过细胞裂解10次粉碎细胞。</li>
	<li>离心机在 20800 x g 15 分钟在4°c 和收集上清入新的1.5 毫升管。样品可以储存在-80 摄氏度的深冰箱里。</li>
	<li>使用可鉴定蛋白试剂盒测定蛋白质浓度, 并将浓度调整为1.0 µg/µL。</li>
	<li>混合5µL 样品和5µL 2x 月桂酸钠 (sds) 样品缓冲器 (10% SDS; 14% 0.5M 三盐酸溶液, ph 6.7; 1% 0.5M EDTA-2Na 溶液, ph 8.0; 20% 甘油; 0.004% 溴酚蓝色染料, 5% β巯基乙醇) 和热在70°c 为10分钟。</li>
	<li>将样品装入4-12% 双三蛋白凝胶中, 在 150 v 上运行1小时。</li>
	<li>在每个缓冲器中浸泡一张厚的滤纸: (浓缩阳极缓冲器: 0.3 米三盐酸, 20% 甲醇; 阳极缓冲: 0.03 米三盐酸, 20% 甲醇; 阴极缓冲器:25 毫米三, 20% 甲醇, 40 毫米 6-氨基 n-己酸, 0.01% SDS)。</li>
	<li>在二十年代将乙烯氟 (PVDF) 膜浸泡在甲醇中, 然后将其转移到阳极缓冲器中。</li>
	<li>平衡的凝胶后, 在 SDS 页与阴极缓冲 5-10 分钟的鼓动下。</li>
	<li>将滤纸、PVDF 膜和凝胶放在半干式印迹机上, 如下所示: 浓缩阳极缓冲纸/阳极缓冲纸/PVDF 膜/蛋白凝胶/阴极缓冲纸 (图 2)。</li>
	<li>覆盖堆栈, 并开始传输在 20 V 30 分钟。</li>
	<li>转移后, 用 PBST (PBS 0.05% 20) 冲洗 PVDF 膜一次。</li>
	<li>在室温下阻断膜30分钟, 在5% 脱脂奶粉中溶解在 PBST 或在4°c 过夜。</li>
	<li>稀释纯化的小鼠抗 SMN 抗体和兔抗 Cofilin 抗体1:10,000 在5% 脱脂奶粉 PBST。在室温下或在4摄氏度的一夜内孵化出1小时的膜。</li>
	<li>用 PBST 洗3次, 10 分钟。</li>
	<li>稀释二次抗体 (HRP 共轭山羊抗鼠和抗兔 IgG (H + L)) 到1:10,000 在5% 脱脂奶粉中 PBST。在室温下在黑暗中孵育1小时。</li>
	<li>用 PBST 3 次冲洗膜10分钟。</li>
	<li>使用化学发光的印迹检测试剂盒检测带信号。</li>
</ol>

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P., 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=Comparing+in+vitro+and+in+vivo+activity+of+2'-O-[2-(methylamino)-2-oxoethyl]-+and+2'-O-methoxyethyl-modified+antisense+oligonucleotides.">Comparing in vitro and in vivo activity of 2'-O-[2-(methylamino)-2-oxoethyl]- and 2'-O-methoxyethyl-modified antisense oligonucleotides.</a> J Med Chem. 51 (9), 2766-2776 (2008).</li><li>Aoki, 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=Bodywide+skipping+of+exons+45-55+in+dystrophic+mdx52+mice+by+systemic+antisense+delivery.">Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery.</a> Proc Natl Acad Sci U S A. 109 (34), 13763-13768 (2012).</li></ol>

作者没有什么可透露的。

这里描述的体外评估方法是快速、容易和敏感的, 足以测试各种 AONs。该协议广泛适用于外显子的跳跃和包含其他基因和各种细胞类型。此外, 大多数的怡人化学 (PMOs 除外) 可以用这种方法转染。AONs 可以调节内源和人工导入基因的前 mRNA 剪接, 并可与携带靶向基因的质粒载体进行联合转染。
该方法的一个关键步骤是, 当 AONs 转染成纤维细胞时, 细胞融合应约 65-80%。对于?...

脊髓肌萎缩 (SMA) 是一种遗传的致命神经肌肉疾病遗传性的常染色体隐性模式。它的特点是运动神经元的退化和渐进躯干和肢体肌肉麻痹1,2。大多数 SMA 的发生是由于在存活的马达神经元 1 (SMN1) 基因3的纯合突变。运动神经元 2 (SMN2) 基因的存活是SMN1的反向复制, 其几乎相同的序列与仅五个基4、5不同。位于外显子7中的SMN2中的 C 到 T 转换使该基因几乎无法正常进行, 因为该突变导致了在几乎90% 的SMN2记录 (图 1a) 中排除的必需的外显子7。SMN2基因缺少的外显子7无法补偿SMN1函数, 因为其蛋白质产品不稳定且快速降解。
反义疗法最近成为治疗 SMA6的非常有前途的策略。美国食品和药物管理局 (FDA) 最近批准了 nusinersen, 使它成为治疗 SMA7的第一种药物。Nusinersen 是一个18的反义寡核苷酸 (怡人) 与-2′-乙修改 (教育部) 和硫代核苷酸骨干。该药物的目标是内含子剪接消声器 N1 (ISS-N1) 位于 SMN2 基因的内含子 7. nusinersen 对 ISS-N1 的约束力通过诱导外显子7包含 (图 1)8,9,10, 促进从内源SMN2基因中恢复功能性全长 SMN 蛋白表达..目前, 对 SMA 治疗的许多研究都集中在研究新的可比 nusinersen 更有效和更少毒性的新型怡得化学药物。已经证明, 与其他化学成分的 AONs 也有效地诱导外显子包含在SMN2显子7两个在体内外和在体内11,12,13。
锁定核酸 (LNAs) 是化学修饰的 RNA 类似物, 包含一个亚甲桥连接 4′--碳与 furanose 结构内的-2′--氧(图 1b)14,15。与 dna 或 rna 相比, LNAs 具有更强的亲和力, 可以结合到互补 RNA 序列中, 并且对内源性核酸具有高度抵抗力。低噪声放大化学已被应用作为探针荧光原位杂交 (鱼) 和在 qPCR16,17。此外, 它被用来调节基因表达的在体外和在体内。GapmeR AONs 是单链 DNA 分子在5′和3′端两侧由几个 LNAs 的组合。它们通过对目标基因进行绑定互补来降低基因表达, 从而使它们被激活的 RNase H18所降解。低噪声 AONs/dna mixmers 是由 LNAs 之间集成的 dna 核苷酸组成的。在这个方向上, 它们可以绑定 miRNA 以抑制其功能 (低噪声 antimiR)19。一些低噪声 antimiRs 已达到临床发展。举例来说, miravirsen (AntimiR-122) 是一个低噪声的 AntimiR, 抑制 miR-122 治疗丙型肝炎感染, 而第二阶段临床试验目前正在进行19,20。最近, 已经证明, 低噪声/DNA mixmers 也可以调制 RNA 拼接21。它可以绑定一个特定序列的 mrna 和诱导外显子跳过肌萎缩蛋白 mrna 和显子包含在SMN2 mRNA在体内13,22.
在本文中, 我们概述了一种方法, 以诱导外显子夹杂使用 AONs 和评价的功效在 RNA 和蛋白质水平。为了举例说明这种方法, 我们在SMN2的内含子7中使用了低噪声/DNA mixmers 靶向 ISS-N1。AONs 转染 SMA 患者细胞的 lipotransfection 诱导外显子7包含在最后的SMN2转录和上调 SMN 蛋白生产。利用低噪声/DNA mixmers 诱导外显子夹杂的优点之一是, 转染的有效浓度明显低于其他化学试剂13。该方法除 phosphorodiamidate 吗啉代寡聚物 (PMOs) 外, 还可用于其他许多 AONs, 由于其中性电荷, 需要通过内-波特共转染试剂诱导的吞进入细胞。

<table border="0" cellpadding="0" cellspacing="0" style="width:655px;" width="655">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SMA Fibroblasts</td>
			<td style="width:153px;">Coriell NIGMS human genetic cell repository</td>
			<td style="width:119px;">GM03813</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">11320033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">F1051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">25300062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serum-deprived media</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">31985070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transfection reagent</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">15338100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">guanidinium thiocyanate-phenol-chloroform (TRIzol)</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">15596-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RT-PCR Primers</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence 5&#39; to 3&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SMN2 forward:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>CTGCCTCCATTTCCTTCTG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SMN2 reverse:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>TGGTGTCATTTAGTGCTGCTC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GAPDH forward:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>TCCCTGAGCTGAACGGGAAG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GAPDH reverse:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>GGAGGAGTTTGGTCGCTGT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">qPCR Primers</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">full-length SMN2 forward:</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>GCTATCATACTGGCTATTATATGGGTTTT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">full-length SMN2 reverse:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>CTCTATGCCAGCATTTCTCCTTAAT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#8710;7&#160;SMN2 forward:</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>TCTGGACCACCAATAATTCCCC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#8710;7&#160;SMN2 reverse:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>ATGCCAGCATTTCCATATAATAGCC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GAPDH forward:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>GCAAATTCCATGGCACCGT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GAPDH reverse:</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>AGGGATCTCGCTCCTGGAA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">P3803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycogen, RNA grade</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">R0551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ Software</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease cocktail inhibitor&#160;</td>
			<td style="width:153px;">Roche</td>
			<td style="width:119px;">11836153001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cathode Buffer</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>0.025 M Tris base + 40&#160;mM 6-aminocaproic acid + 20% Methanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anode Buffer</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>0.03 M Tris Base + 20% Methanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concentred Anode Buffer</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>0.3 M Tris base + 20% Methanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beta Mercaptoethanol&#160;</td>
			<td style="width:153px;">Millipore</td>
			<td style="width:119px;">ES-007-E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVDF membrane&#160;</td>
			<td style="width:153px;">GE</td>
			<td style="width:119px;">10600021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Loading/sample buffer for Western blotting</td>
			<td style="width:153px;">NuPage Invitrogen</td>
			<td style="width:119px;">NP007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">One-Step RT-PCR kit&#160;</td>
			<td style="width:153px;">Qiagen&#160;</td>
			<td style="width:119px;">210210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTPs</td>
			<td style="width:153px;">Clontech</td>
			<td style="width:119px;">3040</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of exon inclusion induced by splice switching antisense oligonucleotides in sma patient fibroblasts

脊髓肌萎缩 (SMA), 一个致命的神经系统疾病造成的损失的SMN1, 提出了一个独特的情况下, 反义寡核苷酸 (怡) 介导治疗领域。虽然SMN1突变负责该疾病, 但已显示在SMN2(包括 FDA 批准的 nusinersen) 中的 AONs 目标内含子拼接消声器 (ISS) 站点已被证明可以恢复 SMN 表达式并改善症状。目前, 对 SMA 治疗的许多研究都侧重于调查以SMN2为目标的新型怡得化学药物, 其效果可能比 nusinersen 更有效、毒性更小。在这里, 我们描述了一个体外的协议, 利用 AONs 的 lipotransfection 进行反向转录聚合酶链反应 (rt-pcr)、定量聚合酶链反应 (qPCR) 和西方印迹的研究, 对显子夹杂物进行评价。这种方法可用于各种类型的安怡化学。使用这种方法, 我们证明由交替锁定核酸 (LNAs) 和 dna 核苷酸 (低噪声/dna mixmers) 组成的 AONs, 导致有效的SMN2外显子包含和恢复 SMN 蛋白在极低浓度, 因此, 放大器/基于 DNA mixmer 的反义寡核苷酸可能是治疗遗传疾病引起的剪接缺损的一种很有吸引力的治疗策略。这里描述的体外评估方法是快速、容易和敏感的, 足以测试各种新颖的 AONs。

利用 SMA 患者成纤维细胞, 我们瞄准了SMN2基因内含子7中的 ISS-N1 消声器区域, 八种不同的反义放大器/DNA mixmers 被转染为 5 nM 浓度 (图 3)。每一个 mixmers 包含一个修改过的 phosphorothioated 骨干, 使他们能够抵御核酸酶退化。为了评估 mixmers 的有效性, 通过 rt-pcr 和 qPCR, 使用SMN2的引物, 量化了SMN2外显子7包含率。外显子7的包含效率从 78-98%...

Watch this Scientific Journal Video about Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts at JoVE.com

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

剪接开关反义寡核苷酸诱导的 SMA 患者成纤维细胞外显子夹杂的评价

各种反义寡核苷酸 (AONs) 被证明可以诱导外显子夹杂 (剪接调制) 和抢救 SMN 表达的脊髓肌萎缩 (SMA)。在这里, 我们描述了一个协议, 以 lipotransfection 诱导外显子包含在SMN2基因和评价方法, 以确定的有效性, SMA 患者成纤维细胞。

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide ...

evaluation-exon-inclusion-induced-splice-switching-antisense

Research

JoVE Journal

Medicine

13.2K 次观看.  University of Alberta Faculty of Medicine and Dentistry. 各种反义寡核苷酸 (AONs) 被证明可以诱导外显子夹杂 (剪接调制) 和抢救 SMN 表达的脊髓肌萎缩 (SMA)。在这里, 我们描述了一个协议, 以 lipotransfection 诱导外显子包含在SMN2基因和评价方法, 以确定的有效性, SMA 患者成纤维细胞。

视频: Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Vascular Occlusion Training for Inclusion Body Myositis: A Novel Therapeutic Approach

Inclusion body myositis (IBM) is a rare idiopathic inflammatory myopathy. It is known to produces remarkable muscle weakness and to greatly compromise function and quality of life. Moreover, clinical practice suggests that, unlike other inflammatory myopathies, the majority of IBM patients are not responsive to treatment with immunosuppressive or immunomodulatory drugs to counteract disease progression1. Additionally, conventional resistance training programs have been proven ineffective in restoring muscle function and muscle mass in these patients2,3. Nevertheless, we have recently observed that restricting muscle blood flow using tourniquet cuffs in association with moderate intensity resistance training in an IBM patient produced a significant gain in muscle mass and function, along with substantial benefits in quality of life4. Thus, a new non-pharmacological approach for IBM patients has been proposed. Herein, we describe the details of a proposed protocol for vascular occlusion associated with a resistance training program for this population.

The present article presents the details pertaining to the application of resistance training associated to vascular occlusion in IBM patients.

血管闭塞包涵体肌炎培训：一种新的治疗方法

Inclusion body myositis (IBM) is a rare idiopathic inflammatory myopathy. It is known to produces remarkable muscle weakness and to greatly compromise ...

科研

医学

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

小鼠模型的腔内MCAO脑梗塞评价甲酚紫染色

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

多巴酚丁胺诱导的心脏压力测试和监控通过在小鼠心脏导管插入术

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype&#160;is&#160;more similar to human DMD patients, was&#160;used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

Exon skipping is currently a&#160;most promising therapeutic option for Duchenne muscular dystrophy (DMD). To expand the applicability for DMD patients and to optimize the stability/function of the resulting truncated dystrophin proteins, a multi-exon skipping approach using cocktail antisense oligonucleotides was developed and we demonstrated systemic dystrophin rescue in a dog model.

多外显子跳跃在犬X连锁肌营养不良用鸡尾酒反义寡核苷酸

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon ...

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

Circumlimbal 缝合法诱导大鼠和小鼠青光眼模型的建立

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

定量 =18F_-Naf-PET-MRI 分析,用于评估面部腰痛患者的动态骨质周转率

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

小鼠右心室系统及缺氧诱发肺动脉高血压的侵入性血液动力学评估

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Characterizing Exon Skipping Efficiency in DMD Patient Samples in Clinical Trials of Antisense Oligonucleotides

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease that causes progressive loss of muscle mass, leading to premature death. The mutations often cause a distorted reading frame and premature stop codons, resulting in an almost total lack of dystrophin protein. The reading frame can be corrected using antisense oligonucleotides (AONs) that induce exon skipping. The morpholino AON viltolarsen (code name: NS-065/NCNP-01) has been shown to induce exon 53 skipping, restoring the reading frame for patients with exon 52 deletions. We recently administered NS-065/NCNP-01 intravenously to DMD patients in an exploratory investigator-initiated, first-in-human trial of NS-065/NCNP-01. In this methods article, we present the molecular characterization of dystrophin expression using Sanger sequencing, RT-PCR, and western blotting in the clinical trial. The characterization of dystrophin expression was fundamental in the study for showing the efficacy since no functional outcome tests were performed.

Here, we present the molecular characterization of dystrophin 38 expression using Sanger sequencing, RT-PCR, and western blotting in the clinical trial.

反义寡核苷酸临床试验中DMD患者样本的Exon跳过效率特征

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease that causes progressive loss of muscle mass, leading to premature death. The mutations ...