Name: in vitro synthesis of modified mrna for induction of protein expression in human cells
Uploaded: 2014-11-13T13:00:00.000+00:00
Duration: 10 min 7 s
Description: Watch this Scientific Journal Video about In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells at JoVE.com

该项目是在巴登 - 符腾堡州，德国资助的欧洲社会基金。

1.增强质粒含有所需的编码DNA序列（CDS） <ol><li>预暖的SOC培养基，其中包括在转化试剂盒，室温并含有100μg/ ml氨苄青霉素至37℃的LB琼脂平板上。平衡的水浴中以42℃。 </li><li>解冻的化学组分E的一个小瓶大肠杆菌在冰上。 </li><li>添加1-5微升质粒（第10至100毫微克）含有CDS成化学成分E.小瓶大肠杆菌 ，轻轻混匀。不要吹打混合细胞。加入质粒后，通过敲击管轻轻拌匀。 </li><li>在冰上孵育混合物30分钟。 </li><li>热休克E.大肠杆菌 30秒在42℃的水浴中不摇动。 </li><li>放置在冰上小瓶2分钟。 </li><li>添加250μl的预热SOC培养基对大肠杆菌大肠杆菌和水平摇晃细菌在300rpm下进行1小时，在37℃的细菌培养箱中培养。 </li><li><html>散布加入100μl和150μl的从转化混合物在预热的LB琼脂平板上，倒置平板孵育过夜，在37℃。 </li><li>后菌落变得可见，接种从各板单菌落到含有5ml的LB培养基中以100μg/ ml的氨苄青霉素的15毫升的培养管中。过夜孵育它们在37℃下，以300rpm振荡培养，直到培养是在晚对数或固定相。 </li><li>对于长期储存的转化的大肠杆菌的大肠杆菌，移液管将75μl100％的甘油，放入离心管。加入225微升的细菌培养（冷冻股票将包含25％甘油）中，拌匀和离心管储存在-80℃。 </li><li>分离含有使用根据制造商的说明书的质粒纯化试剂盒所需的CDS的质粒。 </li><li>测定使用分光光度计质粒浓度。 </li><li>制备的20微升等分试样，并将其存储在-20℃下延长的时间。 </li></ol>插入质粒和_title"&gt; 2。扩增聚合酶链反应加聚T型尾翼（PCR） <ol><li>注:为了获得用于体外转录（IVT）的，感兴趣的CDS的，这里的eGFP，用PCR扩增的DNA模板。同时，聚T型尾翼120胸腺嘧啶（T），通过使用反向引物使用T 120分机加入到插入物。因此，所产生的mRNA得到聚A尾与IVT之后，定义的长度。 </li><li>制备PCR反应混合物中如表1中详述。 </li><li>调匀反应混合物。注:PCR产物可以在4-5，IVT反应中使用。以增加DNA模板量为IVT，PCR混合物的总体积可以增加。几个PCR试管各自含有100微升的PCR混合物可用于获得足够的产率的PCR产物。 </li><li>将PCR管中的热循环仪和用PCR循环方案（ 表2）中运行的PCR。 </li><li>清理P使用根据制造商的说明书和洗脱，用20微升不含核酸酶的水将DNA中的PCR纯化试剂盒CR反应。 </li><li>测量用分光光度计中的DNA的浓度。注:检测DNA浓度是约300微克/毫升。因此，预期的总量应为〜6微克。的DNA的其它蛋白质的量可以根据CDS的长度不同时，用于PCR的退火温度的引物，以及循环数质粒的量。 </li><li>冷冻在-20℃下将DNA很长一段时间，或者直接将其用于IVT。 </li></ol> 3.控制步骤:PCR产物的质量<ol><li>检查PCR产物经DNA凝胶电泳的质量。 </li><li>混合琼脂糖所需量与在烧瓶1×TBE。为1％的琼脂糖凝胶中，添加0.5克琼脂糖至50ml 1×TBE的。 </li><li>微波直到琼脂糖溶解。不要让它沸腾。确保琼脂糖完全在溶液中。 </li><li>添加5μl的核酸凝胶染料至50ml琼脂糖凝胶。 </li><li>倒胶至琼脂糖凝胶中，设置了梳理，并等待大约1小时，直到凝胶固化。 </li><li>取出梳子，并将凝胶中的凝胶托盘。 </li><li>混合的80-10,000 bp的DNA梯2微升（200毫微克）与200纳克的PCR产物的每一个与2μl的6×加样缓冲液中的12微升的总体积。 </li><li>谨慎地装载DNA样品放入琼脂糖凝胶的孔中。 </li><li>使用1×TBE为电泳缓冲液，运行在100伏的琼脂糖凝胶1小时。 </li><li>可视化的成像系统（ 图2）上的DNA条带。 </li></ol> 4. 体外转录（IVT） <ol><li>注:在PCR后，将质粒插入物扩增和聚T形尾序被添加。在IVT中，遗传信息转录的DNA的RNA。所产生的mRNA转录物被用于产生在细胞中的蛋白质。 </li><li>清洁工作区和移液器与核糖核酸酶ðecontamination解决方案。利用PCR洁净和无菌双过滤器枪头，并经常更换手套，以减少污染与RNA酶反应的混合。 </li><li>准备在NTP /帽类似物的混合物，如表3所述。 </li><li>涡旋调匀的NTP /帽模拟混合后短暂离心。 </li><li>组装IVT反应混合物，如表4所述。 </li><li>轻轻上下吹打调匀的IVT反应混合物。然后离心PCR管短暂地在管的底部，收集混合物。 </li><li>在37℃下进行3小时的恒温。 注:最佳孵育时间取决于刀片和定模板的转录效率的长度。对于短插入物（&lt;500 nt）的，较长的温育时间可以是有利的。的时间 - 过程实验可以做到的，以确定最佳孵育时间为最大的产率。因此，设立IVT反应，对于为例即，对1，2，3，4，6小时，并温育过夜，并测定mRNA的量。 在体外转录为代的eGFP基因的动力学显示在图3。 </li><li>要删除的模板DNA，加入1微升的DNA酶（2 U /μL）到IVT反应混合物中，搅拌均匀，孵育15分钟，在37℃。 </li><li>纯化使用根据生产厂家的说明书一RNA纯化试剂盒的反应混合物中。从所述旋转柱的膜用40微升不含核酸酶的水洗脱修饰的mRNA的两倍。注:检测RNA浓度是大约1500微克/毫升。因此，预期的总量应为〜120微克。合成的mRNA的其它蛋白质的量可以不同，取决于CDS的长度和PCR产物的用于IVT的量。 </li></ol> 5.治疗纯化的mRNA与南极磷酸酶<ol><li>注:生成的mRNA与磷酸酶处理，除去5&#39;三磷酸盐，其可以通过RIG-I识别并导致免疫激活。另外，磷酸酶处理防止再环化中的自连接反应。 </li><li>加入9微升10倍南极磷酸酶反应缓冲液79微升纯化的mRNA的解决方案。随后，加入2微升南极磷酸酶（5 U /μL），轻轻混匀。孵育在37℃下将反应混合物搅拌30分钟。 </li><li>纯化使用根据生产厂家的说明书一RNA纯化试剂盒的反应混合物中。从旋转柱膜用50μl无核酸酶的水洗脱修饰的mRNA的两倍。 </li><li>测量用分光光度计改性mRNA的浓度。检查吸收在260nm / 280nm的比值（A 260 / A 280）为≥1.8的260比/ A 230是2.0，这表明纯度。 注:预期总收率应该是大约90微克，这是sufficie新台币约36基因转染。 </li><li>通过加入不含核酸酶的水调整的mRNA至100毫微克/微升的浓度。 </li><li>分装修饰的mRNA成所需的转染，并储存于-80℃下单次使用的等分试样。避免反复冻融循环。正确制备和储存的mRNA稳定多年。在工作​​过程中，始终保持修饰的mRNA在冰上。 </li></ol> 6.控制步骤:合成修饰mRNA的质量<ol><li>查改性mRNA的RNA电泳凝胶的质量。 </li><li>混合琼脂糖所需量与在烧瓶1×TBE。为1％的琼脂糖凝胶中，添加0.5克琼脂糖至50ml 1×TBE的。 </li><li>微波直到琼脂糖溶解。不要让它沸腾。确保琼脂糖完全在溶液中。 </li><li>添加5μl的核酸凝胶染料至50ml琼脂糖凝胶。 </li><li>倒胶成琼脂糖凝胶中，设置了梳理，并等待大约1小时，直到凝胶索利dified。 </li><li>取出梳子，并将凝胶中的凝胶托盘。 </li><li>混合3微升0.5-10 kb的RNA的梯子（3微克）与200纳克的合成的修饰mRNA的每个组中的10微升的总体积7微升加样缓冲液。加样缓冲液的组成在表5中描述。 </li><li>变性梯和mRNA样品在70℃下进行10分钟。 </li><li>谨慎地装载梯并在1％琼脂糖凝胶的mRNA样品。 </li><li> 1小时用1×TBE为电泳缓冲液进行电泳，在100伏。 </li><li>直观上的UV透射和照片使用凝胶记录系统（ 图4）的阶梯和mRNA条带。 </li></ol> 7.制备细胞用于转染<ol><li>板2×10 5个细胞（HEK293细胞）每12孔板井。 </li><li>在细胞培养箱中过夜培养的细胞，在37℃。注:在转染当天，将细胞应具有REAC建置80-90％汇合。 </li></ol> 8.执行细胞基因转染<ol><li>解冻修改后的表达。 </li><li>生成脂质复合物转染。 </li><li>添加25μl的修饰的mRNA（2.5微克）和2微升阳离子脂质转染试剂，以473微升的Opti-MEM（最低必需培养基）予还原的血清培养基中，以产生转染混合物的12孔板的一个孔中的转染。根据要转染井的数量扩展的卷。作为阴性对照，不表达制备转染混合物。 </li><li>通过移液轻轻混合组分。在室温下孵育转染混合物20分钟，以产生脂质复合物进行转染。 注:转染混合物不含抗生素。因此，一定要确保无菌处理，同时细胞。 </li><li>用500微升DPBS洗涤细胞/孔。 </li><li>微升转染混合物加入500一个良好12孔解放军TE。 </li><li>孵育细胞4小时，在37℃和5％的CO 2。 </li><li>吸去转染混合物，并加入1 ml的完整的细胞培养基的细胞中。 </li><li>孵育细胞24小时，在细胞培养箱中培养。 </li><li>进行荧光显微分析使用荧光显微镜（ 图5）。注意:为了检测在细胞中的其它蛋白质，其通过转染产生的与其他的mRNA比的eGFP编码mRNA的表达，该细胞可以培养在盖玻片上。然后将转染能进行和所述细胞可被染色的合适的抗体和通过荧光显微镜进行分析。 </li></ol>在细胞EGFP表达9.流式细胞仪分析<ol><li>从孔中除去细胞培养基，并用1毫升DPBS井洗各（无钙和镁）。加入500μl胰蛋白酶/ EDTA，每孔以从细胞培养板的表面分离的细胞。随后，加入500TNS微升每孔灭活胰蛋白酶。 </li><li>离心分离的细胞5分钟，在300×g离心室温。除去上清液只留下沉淀。重悬在150μl的细胞固定溶液中的细胞团块和细胞悬浮液转移到一个流式细胞管。 </li><li>分析eGFP的表达细胞的百分比，并使用几何平均值（荧光强度的几何平均值）（ 图6）的荧光强度。 </li></ol> 10.测量蛋白表达随着时间的推移<ol><li>作为另外在步骤8中所述的方法进行细胞的3个孔的12孔平板中的mRNA的转染，不加入mRNA的孵育3个孔的HEK293细胞与转染混合物。 </li><li>测量eGFP的1，2的表达，并且在转染后3天，以评估蛋白质表达（ 图7）的持续时间。 </li></ol>

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	<li>Bangel-Ruland, N., 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=CFTR-mRNA+delivery:+a+novel+alternative+for+cystic+fibrosis+&amp;#34;gene+therapy&amp;#34;.">CFTR-mRNA delivery: a novel alternative for cystic fibrosis &amp;#34;gene therapy&amp;#34;.</a> The journal of gene medicine. , (2013).</li><li>Benteyn, D., 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=Design+of+an+Optimized+Wilms&amp;#34;+Tumor+1+(WT1)+mRNA+Construct+for+Enhanced+WT1+Expression+and+Improved+Immunogenicity+In+Vitro+and+In+Vivo.">Design of an Optimized Wilms&amp;#34; Tumor 1 (WT1) mRNA Construct for Enhanced WT1 Expression and Improved Immunogenicity In Vitro and In Vivo.</a> Molecular therapy Nucleic acids. 2, 134 (2013).</li><li>Petsch, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+efficacy+of+in+vitro+synthesized,+specific+mRNA+vaccines+against+influenza+A+virus+infection.">Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection.</a> Nature. 30, 1210-1216 (2012).</li><li>Mandal, P. K., Rossi, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+human+fibroblasts+to+pluripotency+using+modified+mRNA.">Reprogramming human fibroblasts to pluripotency using modified mRNA.</a> Nature protocols. 8, 568-582 (2013).</li><li>Warren, 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=Highly+efficient+reprogramming+to+pluripotency+and+directed+differentiation+of+human+cells+with+synthetic+modified+mRNA.">Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.</a> Cell stem cell. 7, 618-630 (2010).</li><li>Yakubov, E., Rechavi, G., Rozenblatt, S., Givol, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+fibroblasts+to+pluripotent+stem+cells+using+mRNA+of+four+transcription+factors.">Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors.</a> Biochemical and biophysical research communications. 394, 189-193 (2010).</li><li>Anderson, B. R., 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=Incorporation+of+pseudouridine+into+mRNA+enhances+translation+by+diminishing+PKR+activation.">Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation.</a> Nucleic acids research. 38, 5884-5892 (2010).</li><li>Kariko, K., Buckstein, M., Ni, H., Weissman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+RNA+recognition+by+Toll-like+receptors:+the+impact+of+nucleoside+modification+and+the+evolutionary+origin+of+RNA.">Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.</a> Immunity. 23, 165-175 (2005).</li><li>Kariko, 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=Incorporation+of+pseudouridine+into+mRNA+yields+superior+nonimmunogenic+vector+with+increased+translational+capacity+and+biological+stability.">Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.</a> Molecular therapy : the journal of the American Society of Gene Therapy. 16, 1833-1840 (2008).</li><li>Kariko, K., Weissman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+nucleoside+modifications+suppress+the+immunostimulatory+activity+of+RNA:+implication+for+therapeutic+RNA+development.">Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development.</a> Current opinion in drug discovery &amp; development. 10, 523-532 (2007).</li><li>Kormann, M. S., 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=Expression+of+therapeutic+proteins+after+delivery+of+chemically+modified+mRNA+in+mice.">Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.</a> Nature biotechnology. 29, 154-157 (2011).</li><li>Hacein-Bey-Abina, S., 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=Insertional+oncogenesis+in+4+patients+after+retrovirus-mediated+gene+therapy+of+SCID-X1.">Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.</a> The Journal of clinical investigation. 118, 3132-3142 (2008).</li><li>Hacein-Bey-Abina, S., 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=Efficacy+of+gene+therapy+for+X-linked+severe+combined+immunodeficiency.">Efficacy of gene therapy for X-linked severe combined immunodeficiency.</a> The New England journal of medicine. 363, 355-364 (2010).</li><li>Avci-Adali, 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=Optimized+conditions+for+successful+transfection+of+human+endothelial+cells+with+in+vitro+synthesized+and+modified+mRNA+for+induction+of+protein+expression.">Optimized conditions for successful transfection of human endothelial cells with in vitro synthesized and modified mRNA for induction of protein expression.</a> Journal of biological engineering. 8, 8 (2014).</li></ol>

作者宣称，他们有没有竞争的财务权益。

mRNA的疗法在再生医学，治疗的疾病和免疫接种领域的巨大潜力。在这段视频中，我们展示了生产稳定，修饰诱导的细胞蛋白表达的mRNA。使用该协议，其他期望的mRNA可被生成。 在体外合成修饰mRNA的允许细胞的转染具有所需的mRNA诱导靶蛋白的表达。由此，所需的蛋白瞬时表达的生理条件下，直到外源递送的mRNA被完全降解。 在此视频中，绿色荧光蛋白的表达，证实了3天的HEK293细胞的eGFP的mRNA的单次转染后。 mRNA分子编码的其他蛋白有可能导致更短的蛋白质的表达时期。该蛋白的表达减少由于外源递送的mRNA的降解。这种技术对于索姆的。因此，该限制权证申请可能是短暂的诱导蛋白表达。因此，为了维持在细胞中的时间较长蛋白表达，反复递送基因是必需的。虽然，基因转染具有非整合到宿主基因组中，从而防止插入突变和癌症和白血病的发展相比，病毒载体的优势， 在体内转染效率可能低于使用病毒载体。 转染试剂的所需mRNA的浓度和量应该为每个不同的细胞类型14，它们是在目标细胞中的外源递送的mRNA，以产生缺失的蛋白质进行优化。 反复冷冻和mRNA的解冻，应避免以保持所产生的mRNA的稳定性。因此，工作等分试样，可以制备。 PCR和IVT之后，只有一个单一的特定频带应该被检测到。否则，PCR循环数，引物annea灵温度，和/或质粒DNA的量应该优化，以​​获得特定的DNA产物为IVT。此外，该IVT的时间和IVT的DNA模板的量可被优化以获得足够数量的特定长度的mRNA的表达。

在细胞中，转录出信使RNA（mRNA）的与mRNA的下面翻译成所需蛋白质确保细胞的正常功能。遗传性或获得性遗传性疾病可导致合成蛋白质的不足，功能失调而引起严重的疾病。因此，一种新的治疗方法来诱导产生缺失或有缺陷的蛋白质是外源递送合成的修饰基因导入细胞，对于所期望的蛋白质编码。因此，细胞触发合成功能蛋白，它们通常不能生产或自然不会需要。使用这种方法，遗传性疾病可以通过引入基因的被纠正，对于有缺陷或缺失的蛋白1码。 mRNA的治疗也可以用于疫苗接种到synthetize蛋白质抗原，其是由肿瘤细胞或病原体表达的。从而，宿主的免疫系统可被激活，有效地消除肿瘤细胞或防止INFEctions 2,3。此外，近年来，基因被成功地用于产生诱导的多能干细胞（iPS细胞）。为了这个目的，成纤维细胞，转染的mRNA诱导重编程的表达因子4-6，并将其与在再生医学中的巨大潜力转化中的iPSC。 先前，使用常规的mRNA的低稳定性和强免疫原性有关。因此，常规的mRNA的临床应用受到限制。然而，胞苷和尿苷的取代5-甲基胞苷和假尿嘧啶mRNA分子通过Kariko和同事内呈现的mRNA分子稳定的生物流体中，并极大地降低了免疫激活7-10，它现在允许修饰mRNA的临床适用性。 使用合成产生的改性的mRNA，所需基因转录物可以暂时在体内 11交付或体外诱导蛋白表达。所引入的基因是由蜂窝翻译机制在生理条件下转化。由于缺乏整合到宿主细胞基因组相比，病毒的基因治疗载体，肿瘤发生的风险，防止12,13。因此，使用改性合成基因疗法将会得到更好的临床认可的未来。 在这里，我们描述了用于生产改性的mRNA（ 图1），转染细胞的mRNA和蛋白表达的转染细胞中的评价的详细协议。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Cell culture&lt;/strong&gt;</td></tr><tr><td>DMEM, high glucose</td><td>PAA</td><td>E15-009</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>10500</td><td></td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>PAA</td><td>P11-010</td><td>100x</td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>PAA</td><td>M11-004</td><td>200 mM</td></tr><tr><td>DPBS without calcium and magnesium&amp;nbsp;</td><td>PAA</td><td>E15-002</td><td></td></tr><tr><td>0.04% Trypsin / 0.03% EDTA&amp;nbsp;</td><td>Promocell</td><td>C-41020</td><td></td></tr><tr><td>TNS</td><td>Promocell</td><td>C-41120</td><td>Trypsin Neutralizing Solution, 0.05% trypsin inhibitor in 0.1% BSA</td></tr><tr><td>HEK-293 cells</td><td>ATCC</td><td>CRL-1573</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Consumables&lt;/strong&gt;</td></tr><tr><td>Tissue culture plates, 12-well&amp;nbsp;</td><td>Corning</td><td>3512</td><td></td></tr><tr><td>Cell culture flask (75 cm&lt;sup&gt;2&lt;/sup&gt;)&amp;nbsp;</td><td>Corning</td><td>430641</td><td></td></tr><tr><td>DNase-and RNase-free 1.5 ml sterile microcentrifuge tubes&amp;nbsp;</td><td>Eppendorf</td><td>0030 121.589</td><td>Safe-Lock, Biopur</td></tr><tr><td>15 ml conical tubes&amp;nbsp;</td><td>greiner bio-one</td><td>188271</td><td></td></tr><tr><td>PCR clean and sterile epT.I.P.S. dualfilter pipette tips&amp;nbsp;</td><td>Eppendorf</td><td>10 &amp;micro;l: 022491202; 100 &amp;micro;l: 022491237; 1,000 &amp;micro;l: 022491253</td><td></td></tr><tr><td>Cryovial</td><td>greiner bio-one</td><td>122279-128</td><td>Cryo.s&amp;nbsp;</td></tr><tr><td>14 ml polypropylene round bottom tube for bacterial culture&amp;nbsp;</td><td>BD Falcon</td><td>352059</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Plasmid amplification and purification&lt;/strong&gt;</td></tr><tr><td>pcDNA 3.3_eGFP Plasmid&amp;nbsp;</td><td>Addgene</td><td>26822</td><td></td></tr><tr><td>One Shot Top10 chemically component Escherichia coli&amp;nbsp;</td><td>Invitrogen</td><td>C4040-10</td><td></td></tr><tr><td>Sterile water (Ampuwa)</td><td>Fresenius Kabi</td><td>1636071</td><td></td></tr><tr><td>LB medium (Luria/Miller)&amp;nbsp;</td><td>Carl Roth</td><td>X968.1</td><td>Dissolve 25 g L&lt;sup&gt;-1 &lt;/sup&gt;in sterile water.</td></tr><tr><td>LB agar (Luria/Miller)&amp;nbsp;</td><td>Carl Roth</td><td>X969.1</td><td>Dissolve 40 g L&lt;sup&gt;-1 &lt;/sup&gt;in sterile water.</td></tr><tr><td>Ampicillin Ready Made Solution</td><td>Sigma Aldrich</td><td>A5354</td><td>100 mg/ml&amp;nbsp;</td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G2025</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit&amp;nbsp;</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;mRNA production&lt;/strong&gt;</td></tr><tr><td>HotStar HiFidelity Polymerase Kit&amp;nbsp;</td><td>Qiagen</td><td>202602</td><td></td></tr><tr><td>QIAquick PCR Purification Kit&amp;nbsp;</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>MEGAscript T7 Kit&amp;nbsp;</td><td>Life Technologies</td><td>AM1334</td><td></td></tr><tr><td>5-Methylcytidine-5&amp;acute;-triphosphate&amp;nbsp;</td><td>Trilink</td><td>N1014</td><td>5-Methyl-CTP</td></tr><tr><td>Pseudouridine-5&amp;acute;-triphosphate&amp;nbsp;</td><td>Trilink</td><td>N1019</td><td>Pseudo-UTP</td></tr><tr><td>3&amp;acute;-&lt;em&gt;O&lt;/em&gt;-Me-m&lt;sup&gt;7&lt;/sup&gt;G(5&amp;acute;)ppp(5&amp;acute;)G RNA cap structure analog&amp;nbsp;</td><td>New England Biolabs</td><td>S1411L</td><td></td></tr><tr><td>RiboLock RNase Inhibitor</td><td>Thermo Scientific</td><td>EO0381</td><td>40 U/&amp;micro;l&amp;nbsp;</td></tr><tr><td>TURBO DNase</td><td>Life Technologies</td><td>AM1334</td><td>2 U/&amp;micro;l (from MEGAscript T7 Kit)</td></tr><tr><td>Antarctic phosphatase&amp;nbsp;</td><td>New England Biolabs</td><td>MO289S</td><td></td></tr><tr><td>RNeasy mini kit&amp;nbsp;</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>RNaseZap solution&amp;nbsp;</td><td>Life Technologies</td><td>AM9780</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Transfection&lt;/strong&gt;</td></tr><tr><td>Lipofectamine 2000 Transfection Reagent&amp;nbsp;</td><td>Invitrogen</td><td>11668-019</td><td>Cationic lipid transfection reagent</td></tr><tr><td>Opti-MEM I Reduced Serum Media&amp;nbsp;</td><td>Invitrogen</td><td>11058-021</td><td>Improved Minimal Essential Medium (MEM) that allows a reduction of Fetal Bovine Serum (FBS) supplementation&amp;nbsp;</td></tr><tr><td colspan="4">&lt;strong&gt;Gel electrophoresis&lt;/strong&gt;</td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A9539</td><td></td></tr><tr><td>Gelred Nucleic Acid Gel Stain</td><td>Biotium</td><td>41003</td><td>10,000x in water&amp;nbsp;</td></tr><tr><td>peqGOLD Range Mix DNA-Ladder&amp;nbsp;</td><td>Peqlab</td><td>25-2210</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Flow cytometry analyses&lt;/strong&gt;</td></tr><tr><td>CellFIX (1x)</td><td>BD Biosciences</td><td>340181</td><td>10x concentrate&amp;nbsp;</td></tr><tr><td colspan="4">&lt;strong&gt;Primer for insert amplification and poly (T) tail PCR&lt;/strong&gt;</td></tr><tr><td>Forward Primer (HPLC-grade) 10 &amp;micro;M</td><td>Ella Biotech</td><td></td><td>5&amp;acute;-TTGGACCCTCGTAC&lt;br /&gt;AGAAGCTAATACG-3&amp;acute;</td></tr><tr><td>Reverse Primer (HPLC-grade) 10 &amp;micro;M</td><td>Ella Biotech</td><td></td><td>5&amp;acute;- T&lt;sub&gt;120&lt;/sub&gt;-CTTCCTACT&lt;br /&gt;CAGGCTTTATTCAA&lt;br /&gt;AGACCA-3&amp;acute;</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Cell incubator</td><td>Binder</td><td></td><td>CO&lt;sub&gt;2&lt;/sub&gt; (5%) and O&lt;sub&gt;2&lt;/sub&gt; (20%)&amp;nbsp;</td></tr><tr><td>CASY cell counter&amp;nbsp;</td><td>Sch&amp;auml;rfe System</td><td></td><td></td></tr><tr><td>Sterile workbench&amp;nbsp;</td><td>BDK Luft-und Reinraumtecknik GmbH</td><td></td><td></td></tr><tr><td>Bacterial incubator&amp;nbsp;</td><td>Incutec</td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td></td></tr><tr><td>ScanDrop spectrophotometer&amp;nbsp;</td><td>Analytic Jena</td><td></td><td></td></tr><tr><td>PCR thermocycler&amp;nbsp;</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Vortex</td><td>peqlab</td><td></td><td></td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Gel apparatus for electrophoresis&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Gel documentation system&amp;nbsp;</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>FACScan System&amp;nbsp;</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Fluorescence microscope&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>Phase-contrast microscope&amp;nbsp;</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

in vitro synthesis of modified mrna for induction of protein expression in human cells

外源递送的期望的细胞编码合成的信使RNA（mRNA），用于诱导蛋白质合成的具有在再生医学，基础细胞生物学，治疗疾病，和细胞的重编程领域的巨大潜力。在这里，我们描述了一步步的协议，用于生成修改的表达与诱导蛋白表达的减少的免疫激活潜力和增加的稳定性，产生的mRNA的质量控制，细胞的转染mRNA和验证通过流式细胞术。达用的eGFP基因的单一转染后3天，在转染的HEK293细胞中产生绿色荧光蛋白。在这个视频文章中，绿色荧光蛋白的mRNA的合成被描述为一个例子。但是，该过程可以适用于生产其它所需的基因。采用改良的mRNA的合成，细胞可被诱导以瞬时表达所需的蛋白，它们通常不会表达。

使用含有的eGFP的CDS的一个的pCDNA 3.3质粒的修饰的eGFP基因的合成成立（ 图1）。通过PCR扩增插入物和聚T形拖尾之后，以大约1100碱基对长度的清晰条带被检测到（ 图2）。增加了IVT时间增加的mRNA（ 图3）的产量。 IVT之后，一个清晰的mRNA条带大约1100碱基对的长度被检测，对应于绿色荧光蛋白的mRNA的要生产的长度（ 图4）。 所产生的绿色荧光蛋白基因的功能，是由HEK293细胞的转染试验。为了这个目的，转染复合物（脂质复合物）使用阳离子脂质的转染试剂中产生。以每12孔板以及2×10 5个细胞中进行转染。绿色荧光蛋白的细胞中的生产，检测用荧光显微镜在转染后24小时（Figu再5）和流式细胞术（ 图6）。 将HEK293细胞用eGFP的表达。 eGFP的表达，测定转染，以评估细胞蛋白表达的持续时间（ 图7）之后，1，2，和3天。 24小时后，蛋白表达量最高的细胞。量减少1.6倍，每第二天。即使是3天后，所述细胞含有eGFP的。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51943/51943fig1highres.jpg" width="500" /> 图1:修饰的mRNA的生产过程的概述编码DNA序列（CDS），与已知的侧翼序列通过PCR使用特异性引物进行扩增。对PCR产物进行纯化并将所产生的DNA的质量被确定。将mRNA从使用体外转录过程的DNA产物产生。该产品是purifie<html> D和与磷酸酶处理以除去5&#39;-三磷酸。之后的额外的纯化和生成的mRNA的质量控制中，mRNA转染可以执行。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51943/51943fig2highres.jpg" width="500" /> 图2:DNA梯状条带和PCR产物后PCR的DNA产物的分析均在1％琼脂糖凝胶上运行。应检测约为1100 bp的清晰的DNA条带。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51943/51943fig3highres.jpg" width="500" /> 图3: 在体外转录的动力学产生的绿色荧光蛋白的mRNA 1）RNA的梯子和IVT制品2后）0分钟，3）10分钟，4）30分钟后，5）180分钟，6）360分钟，和7） DNA模板单独进行，在1％琼脂糖凝胶上运行。 ve_content"FO:保together.within页="总是"&gt; <img alt="图4" fo:content-width="4in" src="/files/ftp_upload/51943/51943fig4highres.jpg" width="400" /> 图4:IVT RNA梯度和IVT的产品后分析mRNA产物分别在1％琼脂糖凝胶上运行。要检测一个明显的表达条带，约1,100 bp的长度。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51943/51943fig5highres.jpg" width="500" /> 图5:HEK293细胞的绿色荧光蛋白基因转染后24小时荧光显微镜分析。细胞以100倍的放大倍数（A）的相位对比图像。（B）中的细胞以100倍的倍率荧光图像。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51943/51943fig6highres.jpg" width="500" /> 身材6:绿色荧光蛋白表达在HEK293细胞绿色荧光蛋白基因转染后流式细胞仪分析24小时的粉色线代表无转基因细胞和蓝线代表绿色荧光蛋白阳性细胞表达绿色荧光蛋白转染后。绿色荧光蛋白基因转染后，所有测量细胞的93.91％，是积极的几何平均值（荧光强度的几何平均值）为720.16。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51943/51943fig7highres.jpg" width="500" /> 图7:eGFP的表达eGFP的表达在转染后1,2天和3天的流式细胞分析在HEK293细胞中的蛋白表达是mRNA转染后最高24小时。此后，量减少1.6倍，每天（n = 3时）。 表1:PCR混合物的组成。 <table border="0" cellpadding="0" cellspacing="0"宽度="482"&gt; <tbody><tr><td> 部件 </td><td> 终浓度 </td><td> 量（微升） </td></tr><tr><td>正向引物</td><td> 0.7微米</td><td> 7 </td></tr><tr><td>反向引物</td><td> 0.7微米</td><td> 7 </td></tr><tr><td> 5X Q-解决方案</td><td> 1个</td><td> 20 </td></tr><tr><td> 5X HotStar HiFidelity PCR缓冲液</td><td> 1个</td><td> 20 </td></tr><tr><td>质粒DNA </td><td> 50毫微克/ 100微升</td><td>变量</td></tr><tr><td> HotStar HiFidelity DNA聚合酶（2.5 U /μL） </td><td> 2.5单位</td><td> 1 </td></tr><tr><td>不含核酸酶的水</td><td></td><td>变量</td></tr><tr><td>总成交量</td><td></td><td> 100 </td></tr></tbody></table> 表1:PCR混合物的组成。 <table border="0"的cellpadding ="0"CELLSPACING ="0"宽度="449"&gt; <tbody><tr><td></td><td> 循环次数 </td><td> 时间 </td><td> 温度（℃） </td></tr><tr><td>起始变性步骤</td><td> 1 </td><td> 5分钟</td><td> 95 </td></tr><tr><td> 3步循环</td><td rowspan="4" style="vertical-align:middle; text-align:center;"> 2-25 </td><td></td><td></td></tr><tr><td> ·变性</td><td> 45秒</td><td> 95 </td></tr><tr><td> ·退火</td><td> 1分钟</td><td> 55 </td></tr><tr><td> ·扩展</td><td> 1分钟</td><td> 72 </td></tr><tr><td>最终的延伸步骤</td><td> 26 </td><td> 10分钟</td><td> 72 </td></tr><tr><td> PCR循环结束</td><td></td><td>不定</td><td> 4 </td></tr></tbody></table> 表2:PCR循环公关otocol。 <table border="0" cellpadding="0" cellspacing="0" width="615"><tbody><tr><td> 部件 </td><td> 股权集中度（MM） </td><td> 终浓度（MM） </td><td> 体积（微升） </td></tr><tr><td> ATP（从MEGAscript T7套装） </td><td> 75 </td><td> 7.5 </td><td> 4 </td></tr><tr><td> GTP（从MEGAscript T7套装） </td><td> 75 </td><td> 1.875 </td><td> 1 </td></tr><tr><td>我-CTP（从TriLink是） </td><td> 100 </td><td> 7.5 </td><td> 3 </td></tr><tr><td>伪UTP（从TriLink是） </td><td> 100 </td><td> 7.5 </td><td> 3 </td></tr><tr><td> 3&#39;-O-ME-M 7 G（5&#39;），PPP（5&#39;）G帽的RNA结构模拟</td><td> 10 </td><td> 2.5 </td><td> 10 </td></tr><tr><td>总成交量</td><td></td><td></td><td> 23 </td></tr></tbody></table>表3:NTP /帽模拟混合组成。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 部件 </td><td> 终浓度 </td><td> 量（微升） </td></tr><tr><td>不含核酸酶的水</td><td></td><td>变量</td></tr><tr><td>核糖核酸酶抑制剂</td><td> 40单位</td><td> 1 </td></tr><tr><td> NTP /帽模拟混合（步骤4.3） </td><td></td><td> 23 </td></tr><tr><td> PCR产物</td><td> 1微克</td><td>变量</td></tr><tr><td> 10×反应缓冲液</td><td> 1个</td><td> 4 </td></tr><tr><td> 10×T7 RNA聚合酶的酶混合物</td><td> 1个</td><td> 4 </td></tr><tr><td>总成交量</td><td></td><td> 40 </td></tr></tbody></table> 表4: 体外转录的组成（我VT）的反应混合物中。 <table border="0" cellpadding="0" cellspacing="0" width="424"><tbody><tr><td> 部件 </td><td> 量（微升） </td></tr><tr><td>甲酰胺</td><td> 3.3 </td></tr><tr><td> 37％的甲醛</td><td> 1 </td></tr><tr><td> MEN缓冲液（10X） </td><td> 1 </td></tr><tr><td> 6X上样缓冲液（用peqGOLD范围混合DNA梯提供） </td><td> 1.7 </td></tr><tr><td>总成交量</td><td> 7 </td></tr></tbody></table> 表5:用于RNA凝胶电泳制备的加样缓冲液。 <table border="0" cellpadding="0" cellspacing="0" width="538"><tbody><tr><td></td><td> 细胞培养液和缓冲液 </td></tr><tr><td> HEK-293细胞培养液中</td><td>添加25毫升的FCS，2.5 ml青霉素/链霉素的2.5毫升的L-谷氨酸的tamine在220毫升DMEM高糖。储存在4℃的培养基，并用它在2周内。 </td></tr><tr><td> TBE缓冲液（10X） </td><td>溶解0.9M的Tris碱，将0.9M硼酸和20mM EDTA的1升水中（Ampuwa）。所述缓冲液的pH为8。 </td></tr><tr><td> MEN缓冲液（10X） </td><td>溶解的200mM MOPS，50mM的醋酸钠，10mM EDTA的在1L水（Ampuwa）。 pH值用NaOH调节至7。 </td></tr></tbody></table> 表6:细胞培养基和缓冲液。

Watch this Scientific Journal Video about In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells at JoVE.com

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

在这个视频文章中，我们描述了诱导细胞蛋白表达的体外合成修饰的mRNA。

在体外合成在人体细胞修饰蛋白表达的mRNA的感应

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

in-vitro-synthesis-modified-mrna-for-induction-protein-expression

Universidad San Sebastian

Research

JoVE Journal

Biology

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

24.9K 次观看.  University Hospital Tuebingen. 在这个视频文章中，我们描述了诱导细胞蛋白表达的体外合成修饰的mRNA。 

视频: In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3&#697; end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5&#697; untranslated region (5&#697; UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.

基于细胞的 sineup 非编码 rna 检测, 可专门增强 mrna 的翻译能力

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

科研

遗传学

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

发育生物学

Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1&#936;-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.

The protocol presents in vitro transcription (IVT) of chemically modified mRNA, cationic liposome preparation, and functional analysis of liposome enabled mRNA transfections in mammalian cells.

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic ...

生物化学

<meta charset="utf8"></head><body>用于微区室化合成细胞的 CFPS 方案

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

<meta charset="utf8"></head><body>作者聚焦：优化 CFPS 系统以进行合成细胞构建

This protocol describes the Cell-Free Protein Synthesis (CFPS) system used in constructing synthetic cells. It outlines key stages with representative results in different micro-compartments. The protocol aims to establish best practices for diverse laboratories in the synthetic cell community, advancing progress in synthetic cell development.

用于构建合成细胞的无细胞蛋白质合成系统

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

阳离子纳米脂质体的生成, 用于高效输送体外转录信使 rna

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

生物工程

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

生物学

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

具有体外转录mRNA的初级巨噬细胞的高效转染

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

免疫与感染

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp&#160;encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp&#160;is produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNp&#160;is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNp&#160;nanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

A self-assembled peptide-poloxamine nanoparticle (PP-sNp) is developed using a microfluidic mixing device to encapsulate and deliver in vitro transcribed messenger RNA. The described mRNA/PP-sNp could efficiently transfect cultured cells in vitro.

使用肽-泊洛沙明纳米颗粒在培养细胞中高效转染 体外 转录的mRNA

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. ...

在体外合成在人体细胞修饰蛋白表达的mRNA的感应

摘要

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阳离子纳米脂质体的生成, 用于高效输送体外转录信使 rna

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