这项工作是由加拿大国家科学与工程研究理事会（NSERC）（＃298527）（BB）的资助。 CT是由一个机构FARE奖学金支持。 AV是由NSERC·格雷厄姆·贝尔博士支持奖学金。 BB召开了加拿大研究主席在人类反转录病毒学（第2层）。由于碧翠丝Beisner在修改文本的帮助。

该UQAM伦理委员会已经批准了这些协议，这是按照大学中心医院，蒙特利尔的圣 - 吕克医院（蒙特利尔，加拿大）的伦理委员会的指导方针。与会者签署了知情同意书。 1.介质制备工艺和主代绒毛滋养细胞的分离<ol><li>通过补充的Dulbecco氏改良的Eagle氏培养基（DMEM）用25mM HEPES，10％胎牛血清（FBS）和1％青霉素/链霉素制备用于人原绒毛细胞滋养细胞的培养基。过滤通过无菌0.22微米的过滤器，并存储所制备的介质在-20℃在50毫升分装℃冰箱。 </li><li>隔离从新鲜胎盘以下标准协议3个主要代绒毛滋养细胞。 <ol><li>用钳子，轻轻挠它关闭删除该组织胎膜，同时小心不要去除绒毛组织。切在使用手术刀和剪刀约3×3厘米的立方体剩余的胎盘绒毛。彻底含0.9％的NaCl除去母体血液清水洗净。 <ol><li>持用钳子每个立方体，小心翼翼地从剩下的血管和纤维组织用剪刀去除绒毛组织。 </li><li>为15至30毫克的绒毛组织，消化四倍的Hank氏平衡盐溶液（HBSS）包含胰蛋白酶（从9.6×10 5〜1.8×10 6 U;终浓度为1.2mg / ml）和脱氧核糖核酸酶I（DNA酶I）（最终浓度200微克/毫升），用于在连续摇动的水浴在37℃下30分钟。 注意:使用150毫升总体积为第一消化，将100ml用于第二消化和75ml对剩下的两个消化。 </li></ol></li><li>收集含有分散细胞，离心上清在1250 XG为不带刹车15分钟。在1毫升预热DMEM含有去除上清和悬浮细胞1％青霉素/链霉素。 </li><li>层上的不连续的5％的顶部的分散的细胞- 70％聚乙烯吡咯烷酮包覆二氧化硅梯度（见梯度制备表1）和离心机在507 xg离心23分钟，不制动。通过抽吸除去1.017之间的密度层以1.033和收集40％和50％之间的各层中使用新的吸管的梯度。用10ml预热的DMEM含有1％青霉素/链霉素洗涤细胞广泛。 注意:将收集的馏分对应密度1.048 1.062和，其中港口之间的滋养层。 </li><li>算使用血球细胞。简单地说，混合细胞，并在新的离心管中加入10微升。加10μl台盼蓝并轻轻地再次混合。绘制出细胞悬液，并填补了血细胞计数室。算下10X目标细胞。 </li><li>放置1×10 6和4.5×10 6细胞到用于分离的试管中的部分1.3和2中，各自LY。种子的剩余的细胞以1.5×10 6个细胞/孔在补充的DMEM培养基和培养物的密度在37℃和5％CO 2的最多4天的其它实验。 </li></ol></li><li>孤立的主要代绒毛滋养细胞的纯度评价<ol><li>自旋为1×10的离心管在300×克新鲜分离的细胞滋养细胞的6。弃上清，加入1ml预热的磷酸盐缓冲盐水（PBS）的。离心机的细胞在300 XG，并通过抽吸去除PBS。 </li><li> 1毫升冷甲醇添加到细胞中并在-20℃下孵育20分钟。离心细胞，在300×g离心5分钟，并通过抽吸除去甲醇。 注意:使用面膜罐，护目镜和橡胶手套在处理甲醇。 </li><li>加入1毫升的PBS在室温下再水合的细胞，离心分离，在300 xg离心并通过抽吸除去PBS中。 </li><li>孵育含有FBS（1:50 [体积/体积]稀释）和一个的FcR的细胞在PBS中在室温下阻断45分钟试剂（1:10），以消除非特异性结合。 </li><li>用500μl室温的PBS，离心细胞洗两次，在300×g离心5分钟，并通过抽吸除去PBS中。悬浮细胞在100μl室温PBS中。 </li><li>孵育细胞与小鼠单克隆异硫氰酸荧光素（FITC）缀合的抗人细胞角蛋白-7抗体克隆LP5K（1:200）或在PBS中的对照同种型匹配的非特异性抗体含有0.2％BSA的45分钟，在室温下黑暗。洗涤细胞在PBS中。 </li><li>通过流式细胞术3分析细胞。使用细胞制剂，其通过流式细胞术进一步的实验表明最低96％的CK-7阳性细胞。 </li></ol></li></ol> 2.人力小学代绒毛滋养细胞的转染<ol><li>使用微孔设备的人原代绒毛滋养细胞的转染<ol><li>制备含有6孔板2ml中微穿孔细胞的培养补充的DMEM培养基中。 </li><li>主细胞滋养细胞（见1.2节）的分离后，取细胞的等分试样中的悬浮液，并使用血细胞计数器确定细胞数。 </li><li>在300 XG转移1.5×10 6细胞到微量离心管中并离心沉淀细胞，在室温下5分钟。清洗细胞用1ml的Dulbecco PBS（DPBS）（Mg 2+离子 ，无Ca 2+）中并在300 xg离心离心沉淀细胞，在室温下5分钟。 </li><li>除去上清液并轻轻重悬细胞于100μl重悬浮缓冲液中。 注意:避免保持细胞悬浮液，在室温下超过15-30分钟，以最大限度地提高细胞生存力和转染效率。 </li><li>加合胞素-2的siRNA（或控制加扰的siRNA）的300毫微克至1.5 ml离心管，并用100微升转染吸管吸出细胞的siRNA混合物。插吸管入站（见材料清单）和邮件主题单元为30毫秒的1300 V单脉冲。 </li><li>立即将细胞转移到含有预热的37℃补充的培养基，以避免细胞损伤的6孔板（在步骤2.1.1的方法制备）。 </li><li>重复步骤其余样品2.1.5和2.1.6。 </li><li>轻轻摇动平板30秒，以确保即使在细胞的分布。孵育在37℃的板在加湿的 CO 2培养箱中培养24至48小时。刷新培养基在转染后24小时。 </li><li>通过评估Western blot分析转染效率（参见步骤3到6）。 </li></ol></li><li>在人类细胞滋养细胞主要的siRNA脂质体<ol><li>绒毛细胞滋养细胞的分离后，种子的细胞以在2ml培养基中每孔1.5×10 6个细胞在6孔板的密度，孵育18小时。 </li><li>稀释合胞-2特异性siRNA的300纳克（或控制加扰RNA干扰）和5μl基于脂质的转染试剂的单独1.5毫升管中的10微升抗生素和无血清培养基的总体积。孵育5分钟。 </li><li>混合经稀释的试剂，并在室温下孵育另外的20分钟，以形成转染复合物。添加90微升抗生素和无血清培养基的到混合至100μl的终体积。 </li><li>从6孔板（步骤2.2.1）除去培养基。加入2毫升的新鲜培养基中，并在100微升转染混合物到每个孔中。孵育在37℃下将细胞在CO 2培养箱中24至48小时。刷新培养基在转染后24小时。 </li><li>通过评估Western blot分析转染效率（参见步骤3到6）。 </li></ol></li></ol>从全细胞裂解液培养3.制备<ol><li>取出介质和冲洗每口井（从步骤2.1.8和2.2.4）采用预热DPBS（镁离子 ，钙离子免费）。吸出DPBS，加入500微升的0.25％胰蛋白酶/乙二胺四乙酸（EDTA）。在CO 2培养箱中孵育1-3分钟，在37℃。通过加入500微升含血清的生长培养基的停止胰蛋白酶处理。 注:按瓶/皿的类型使用适当的介质卷:1毫升，每107 个细胞/ 100mm皿/ 150 平方厘米瓶; 0.5毫升每5×10 6个细胞/ 60mm培养皿/ 75cm 2的烧瓶中。 </li><li>转移细胞到1.5ml微量离心管中，沉淀细胞，在300×g离心在室温下2分钟离心。离心300 xg离心清洗细胞用DPBS（镁离子 ，无Ca 2+）和沉淀的细胞在室温下2分钟。 </li><li>小心取出上清液，而不会干扰颗粒。放置在冰上的管中。重悬沉淀50-200微升冰冷无线电免疫沉淀测定（RIPA）缓冲液（ 见表2缓冲组合物）含有新鲜添加编蛋白酶和磷酸酶抑制剂在1倍终浓度。简单地说，涡管，在冰上留下30分钟。 </li><li>离心16,000 XG 20分钟，在4℃。小心地将管冰上。使用移液管，将上清液转移到新鲜的预冷却的离心管保持在冰上。弃沉淀。 </li></ol>从培养细胞的核提取物的制备4 <ol><li>每口井（步骤1.2.5），洗细胞滋养细胞两次用1毫升预热DPBS（镁离子 ，钙离子免费）的。吸DPBS，加入500微升0.25％胰蛋白酶/ EDTA的。在CO 2培养箱中孵育1-3分钟，在37℃，并通过加入500微升含血清的生长培养基的停止胰蛋白酶处理。 </li><li>转移细胞到1.5ml微量离心管中，沉淀细胞，在300×g离心在室温下2分钟离心。通过离心洗细胞用DPBS（镁离子 ，无Ca 2+）和颗粒细胞在300×g离心在室温下2分钟。仔细清除所有上清而不破坏颗粒，然后将管冰上。 </li><li>根据制造商的说明提取核蛋白。确定使用蛋白定量测定（布拉德福德或双金鸡宁酸测定法（BCA））各样品的蛋白质浓度。 </li></ol> 5.样品制备和电泳<ol><li>确定用于使用蛋白质定量测定各细胞提取物中的蛋白质浓度（例如，布拉德福德8或BCA 9）。 </li><li>根据制造商的说明（如果抗体可在还原和变性条件下使用），2×Laemmli样品缓冲液（ 表2）的等体积的增加总蛋白的20-30微克。 </li><li>煮沸细胞溶胞产物在100℃下5分钟。等分试样50-100微升裂解物，以避免重复的冷冻/解冻循环和剩余的管储存于-20℃以备将来使用的。 </LI&gt; <li>而将样品加热，制备1升1倍的运行缓冲液（ 表2）。 </li><li>旋转样品以16,000 xg离心几秒钟，将它们放置在冰上。 </li><li>加载蛋白质的等量成12％的SDS-PAGE凝胶上（ 见表2为凝胶组合物）的各孔中，用分子量标记一起。在100 V.运行1凝胶2小时</li></ol> 6.从凝胶转移的蛋白质的膜<ol><li>激活用甲醇聚偏二氟乙烯（PVDF）膜1分钟，并用1×转移缓冲液/ 10％甲醇溶液冲洗。根据制造商的说明，进行30分钟依赖于蛋白质大小转移至1小时。 注意:转印效率可使用丽春红染色的封闭步骤之前进行评估。 </li></ol> 7.抗体染色<ol><li>在室温下1小时或过夜，在4℃下，用5％封闭溶液阻断膜。 </li><li> 6或用抗甘油醛-3-磷酸脱氢酶（GAPDH; 0.4微克/毫升; 1:500），在5％封闭溶液过夜:用抗合胞素-2抗体（5000 1 4微克/毫升）孵育膜4℃（抗合胞-2）或在室温下45分钟。三次洗膜在Tris缓冲盐水吐温（TBST; 见表2缓冲组合物）5分钟。 </li><li>孵育适当辣根过氧化物酶（HRP）的膜缀合的抗兔或抗小鼠抗体（1:10,000），在5％在室温下1小时阻断在TBST缓冲液中。洗膜在TBST中3次5分钟，然后在TBS中漂洗。检测使用基于化学发光印迹底信号。 </li></ol> 8.单链寡核苷酸的放射性标记<ol><li>磷酸化反应<ol><li>在微量离心管中，加入50毫微克的正向寡核苷酸一起用1×T4多核苷酸激酶缓冲液，1UT4多核苷酸激酶和20微居里（γ-32 P）ATP和弥补无核酸酶的水反应体积为20微升。 </li><li>孵育在37℃下30分钟。停止加入5微升的0.5M EDTA的反应。补足体积至50μl无核酸酶的水。 </li></ol></li><li>由寡核苷酸未掺入的核苷酸的去除<ol><li>制备具有下列制造商的说明TE缓冲液（ 表2）平衡过的G-25柱。放置在一个新鲜的无DNase的1.5 ml离心管色谱柱。 </li><li>慢慢加入探针（从步骤8.1.2）的50微升过树脂，小心不要去打扰树脂床。自旋2分钟，在350 xg离心收集在1.5 ml离心管中纯化的样品。在350 xg离心洗用36微升不含核酸酶的水和自旋的G-25柱2分钟以收集它在离心管中。 </li></ol></li><li>杂交用互补链醇igonucleotide <ol><li>放射性标记的探针（86微升）转移到1.5ml微量离心管中，并添加反向寡核苷酸的200毫微克与退火缓冲液的1倍（ 见表2）。热，在95℃5分钟并在室温下留下过夜冷却。 </li><li>通过向前加入50毫微克的未标记的制备非放射性的双链寡核苷酸和反向寡核苷酸以及退火缓冲液1×在1.5ml微量离心管中。补足体积至100μl无核酸酶的水。热火并保持在室温下按步骤8.3.1所述。 </li></ol></li></ol> 9.准备一个非变性聚丙烯酰胺凝胶的EMSA <ol><li>用1.5毫米的空间梳（ 见表2为凝胶组合物）制备的4％非变性凝胶（10×12厘米）。清洁用蒸馏水所有玻璃板。 注意:这是至关重要的板完全不含离子洗涤剂，如SDS。 </li><li>马上p我们在板之间的丙烯酰胺溶液允许聚合进行（约30分钟）。 </li><li>组装电泳装置和填充0.5X TBE坦克。在150V样品装载之前预先进行30分钟的凝胶到1小时。 </li></ol> 10. DNA结合反应<ol><li>在1.5ml微量离心管中，在1×凝胶移位结合缓冲液（ 表2）中添加核提取（步骤4.3）的10微克和补足终体积为10微升用无核酸酶水。作为对照，准备一管有没有核提取物。对于竞争反应，加1μL冷非特异性或双链寡核苷酸（步骤8.3.2）的。 </li><li>从步骤8.3.1到每个反应加入1μl放射性探头。在室温下孵育20分钟进行反应。加入1μl的10倍的凝胶上样缓冲液的每反应和加载部分9中制备的凝胶上的每个样品。 </li><li>在15运行1 1/2凝胶至2小时0 V迁移后，包装在塑料包装和定位胶和玻璃下面荧光屏曝光磁带。在4℃离开盒24小时。 </li><li>从曝光纸盒中取出屏幕，并在扫描荧光成像装置插入。 </li></ol>

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K., 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150, 76-85 (1985).</li><li>Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+reaction+patterns+in+first+trimester+placenta:+implications+for+trophoblast+isolation+and+purity+screening.">Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening.</a> Placenta. 21, 733-741 (2000).</li><li>Desforges, M., 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=The+SNAT4+isoform+of+the+system+A+amino+acid+transporter+is+functional+in+human+placental+microvillous+plasma+membrane.">The SNAT4 isoform of the system A amino acid transporter is functional in human placental microvillous plasma membrane.</a> J. Physiol. 587, 61-72 (2009).</li><li>Felgner, P. L., 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=Lipofection:+a+highly+efficient,+lipid-mediated+DNA-transfection+procedure.">Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.</a> Proc Natl Acad Sci U S A. 84, 7413-7417 (1987).</li><li>Guilbert, L. J., 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=Preparation+and+functional+characterization+of+villous+cytotrophoblasts+free+of+syncytial+fragments.">Preparation and functional characterization of villous cytotrophoblasts free of syncytial fragments.</a> Placenta. 23, 175-183 (2002).</li><li>Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+trophoblast+cells.">Isolation and culture of term human trophoblast cells.</a> Methods Mol. Med. 121, 203-217 (2006).</li><li>Ma, B., Zhang, S., Jiang, H., Zhao, B., Lv, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoplex+morphologies+and+their+influences+on+transfection+efficiency+in+gene+delivery.">Lipoplex morphologies and their influences on transfection efficiency in gene delivery.</a> J. Control. Release. 123, 184-194 (2007).</li><li>Freeley, M., Long, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+siRNA+delivery+to+T-cells:+potential+clinical+applications+for+inflammatory+disease,+cancer+and+infection.">Advances in siRNA delivery to T-cells: potential clinical applications for inflammatory disease, cancer and infection.</a> Biochem. J. 455, 133-147 (2013).</li></ol>

The authors have nothing to disclose.

研究人类胎盘功能和发展已通过旨在优化各种胎盘细胞群的分离方案有了很大的提高。其中最好的研究的胎盘细胞群仍是代绒毛滋养细胞，其研究有很大的优化，从许可协议的高效，可靠的隔离受益。这进一步允许了若干实验，如转染和促进剂的研究。使用先前描述的方案3中 ，可以得到主绒毛细胞滋养细胞的纯群体。 CK-7是一种中间丝蛋白，其在滋养细胞中表达。针对此蛋白的单克隆抗?...

Human placental dysfunction is associated with the development of several pregnancy-associated diseases like preeclampsia and intrauterine growth restriction 1. An important cell constituent of the placenta is the trophoblasts, which can be classified as either extravillous or villous cytotrophoblasts. Upon fusion, villous cytotrophoblasts further differentiate into the syncytiotrophoblast layer, a multinuclear cell structure with an important role in feto-maternal exchange and hormone production 2. Human primary villous cytotrophoblasts and their differentiated counterparts represent important biological samples and allow researchers to study a number of placenta-related processes, such as cell fusion, in culture. Furthermore, substantial efforts are ongoing to identify markers that will facilitate appropriate management and improve preventive therapies specific to pregnancy-related diseases. Laboratories routinely isolate primary human villous cytotrophoblasts from fresh placentas, using a standard isolation procedure based on trypsin digestion of placenta villi 3. As cultured cytotrophoblasts lose their capacity to proliferate and quickly differentiate in a syncytiotrophoblast-like layer upon culture 4, very efficient transfection methods and optimized analysis approaches are needed. Previous studies have determined optimal conditions of transfection of these primary cytotrophoblasts 5. Herein, a different method of siRNA transfection, which has been previously tested in this cell type 6, is presented. In comparison to a lipofection-based approach, this microporation method improves transfection, as assessed by the extent of silencing of specific genes.
Promoter and gene expression studies also provide a better understanding of placental function. Although more difficult to use owing to the short time frame for which primary villous cytotrophoblasts can be cultured, promoter analyses using standard protocols can nonetheless be addressed, as previously published 7. Electrophoretic Mobility Shift Assay (EMSA) is one of these commonly used in vitro methods, allowing for fast and easy monitoring of DNA-protein interactions. Nuclear extracts from these primary trophoblasts were used to test a region of the Syncytin-2 promoter for specific interactions. Results revealed that bound factors could be detected at different time points of culture and in a specific and reproducible manner.
Data presented in this protocol confirm that our transfection approach and the EMSA protocol can be used for isolated primary villous cytotrophoblasts and will be of great use to study the diverse functions of villous cytotrophoblasts in normal or pathological conditions.

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sirna transfection and emsa analyses on freshly isolated human villous cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

从足月妊娠胎盘新鲜用于分离人主要代绒毛滋养细胞进行的一组中的协议部分提供实验。继他们的孤立，我们首先分析细胞滋养细胞的纯度通过使用角蛋白7标记（ 图1）。使用单克隆抗细胞角蛋白7的抗体的细胞制备物从而染色。 图1表示从以下主要绒毛细胞滋养细胞的纯化典型的实验中，通过标准的流式细胞仪分析的结果。以下的密度梯度步骤，...

Watch this Scientific Journal Video about siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts at JoVE.com

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

这个协议描述了使用微孔化有效转染的siRNA在新鲜分离的人绒毛细胞滋养细胞和鉴定在这些细胞中DNA的蛋白质复合物的方法。转染的细胞可以通过Western印迹进行监测，并在为期4天的培养时间，EMSA分析。

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

sirna-transfection-emsa-analyses-on-freshly-isolated-human-villous

Research

JoVE Journal

Genetics

10.5K 次观看.  University of Quebec in Montreal. 这个协议描述了使用微孔化有效转染的siRNA在新鲜分离的人绒毛细胞滋养细胞和鉴定在这些细胞中DNA的蛋白质复合物的方法。转染的细胞可以通过Western印迹进行监测，并在为期4天的培养时间，EMSA分析。 

视频: siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

科研

遗传学

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

激光局部补骨脂素加合物的单分子分析

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

重组腺相关病毒9型 MicroRNA 系统分娩治疗啮齿动物神经肌肉疾病

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

基于质谱的蛋白质组学分析, 利用 OpenProt 数据库揭开从非规范开放阅读框架翻译的新蛋白质

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

从小鼠横纹肌瘤分离的原发性肿瘤细胞的肿瘤球衍生和治疗

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

通过全基因组互惠性分析,对糖母菌菌种间热耐性差异的遗传图谱

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

通过心肌毒素损伤和在 Vivo 中注射自传递 siRNA 在骨骼肌再生过程中的卫星细胞功能分析

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...