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><tbody><tr><td>SMA Fibroblasts</td><td>Coriell NIGMS human genetic cell repository</td><td>GM03813</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher</td><td>11320033</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F1051</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher</td><td>15140122</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Thermo Fisher</td><td>25300062</td><td></td></tr><tr><td>Serum-deprived media</td><td>Thermo Fisher</td><td>31985070</td><td></td></tr><tr><td>Transfection reagent</td><td>Thermo Fisher</td><td>15338100</td><td></td></tr><tr><td>guanidinium thiocyanate-phenol-chloroform (TRIzol)</td><td>Thermo Fisher</td><td>15596-018</td><td></td></tr><tr><td>RT-PCR Primers</td><td></td><td></td><td>Sequence 5&#039; to 3&#039;</td></tr><tr><td>SMN2 forward:&amp;nbsp;</td><td></td><td></td><td>CTGCCTCCATTTCCTTCTG</td></tr><tr><td>SMN2 reverse:&amp;nbsp;</td><td></td><td></td><td>TGGTGTCATTTAGTGCTGCTC</td></tr><tr><td>GAPDH forward:&amp;nbsp;</td><td></td><td></td><td>TCCCTGAGCTGAACGGGAAG</td></tr><tr><td>GAPDH reverse:&amp;nbsp;</td><td></td><td></td><td>GGAGGAGTTTGGTCGCTGT</td></tr><tr><td>qPCR Primers</td><td></td><td></td><td></td></tr><tr><td>full-length SMN2 forward:</td><td></td><td></td><td>GCTATCATACTGGCTATTATATGGGTTTT</td></tr><tr><td>full-length SMN2 reverse:&amp;nbsp;</td><td></td><td></td><td>CTCTATGCCAGCATTTCTCCTTAAT</td></tr><tr><td>GAPDH forward:&amp;nbsp;</td><td></td><td></td><td>GCAAATTCCATGGCACCGT</td></tr><tr><td>GAPDH reverse:</td><td></td><td></td><td>AGGGATCTCGCTCCTGGAA</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>P3803</td><td></td></tr><tr><td>Glycogen, RNA grade</td><td>Thermo Fisher</td><td>R0551</td><td></td></tr><tr><td>ImageJ Software</td><td></td><td></td><td></td></tr><tr><td>Protease cocktail inhibitor&amp;nbsp;</td><td>Roche</td><td>11836153001</td><td></td></tr><tr><td>Cathode Buffer</td><td></td><td></td><td>0.025 M Tris base + 40&amp;nbsp;mM 6-aminocaproic acid + 20% Methanol</td></tr><tr><td>Anode Buffer</td><td></td><td></td><td>0.03 M Tris Base + 20% Methanol</td></tr><tr><td>Concentred Anode Buffer</td><td></td><td></td><td>0.3 M Tris base + 20% Methanol</td></tr><tr><td>Beta Mercaptoethanol&amp;nbsp;</td><td>Millipore</td><td>ES-007-E</td><td></td></tr><tr><td>PVDF membrane&amp;nbsp;</td><td>GE</td><td>10600021</td><td></td></tr><tr><td>Loading/sample buffer for Western blotting</td><td>NuPage Invitrogen</td><td>NP007</td><td></td></tr><tr><td>One-Step RT-PCR kit&amp;nbsp;</td><td>Qiagen&amp;nbsp;</td><td>210210</td><td></td></tr><tr><td>dNTPs</td><td>Clontech</td><td>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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

13.3K 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

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

合并绝对和相对定量PCR数据来量化STAT3剪接变体成绩单

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

科研

遗传学

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

利用RNA测序来检测新剪接变异体在相关耐药<em&gt;体外</em&gt;癌症模型

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

癌症研究

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

工程人为因素来特异性地操纵人细胞中的选择性剪接

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

将人类成纤维细胞直接重新编程到肌细胞中，以研究神经肌肉疾病的治疗方法

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

使用 E1A 微型工具研究 mRNA 拼接更改

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

ACT1-CUP1测定确定出芽酵母中剪接体突变体的底物特异性敏感性

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

一种基于报告基因的细胞检测，用于监测剪接效率

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

生物化学

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 ...