dsRNA细胞转染(Entranster)操作步骤
原代细胞转染步骤
原代细胞转染步骤原代细胞转染是一种将DNA、RNA或蛋白质等生物分子导入细胞内的方法，它被广泛应用于细胞遗传和药物开发等领域。
在进行原代细胞转染时，需要仔细的操作并遵循一系列的步骤。
首先，我们需要准备好原代细胞和转染试剂。
原代细胞应当在生长期且处于状态好的状态，如要进行转染的细胞来自动物体内，则需要通过适当的方法进行分离和培养。
转染试剂可以是多种选择，例如：化学试剂、聚合物或与病毒相关的转染试剂。
在选择试剂时，需要考虑到细胞种类和所需转移物之间的互补性和相容性。
接下来，我们需要确定转染试剂的浓度。
过高的浓度可能会导致细胞死亡，而过低的浓度则会降低转染的效果。
因此，应该先进行不同浓度的试验，最后选择最适合的浓度。
然后，我们需要按照试剂提供的说明书或参考文献的指导操作。
通常试剂需要预先混合，试剂的添加需要分几步进行，并在每一步后轻轻的混合。
此外，由于细胞对试剂的反应不同，因此我们需要在不同的细胞类型中进行试验，以找到最佳的处理方式。
接下来，我们需要将转染试剂与细胞混合。
这需要轻轻拍打培养皿或使用转染通道将混合物加入细胞培养基中。
随着细胞被转染并进入生长周期，原代细胞从之前的瞬间转化为可以被恒定表达的细胞。
最后，在细胞与试剂混合后，我们必须为其提供适当的环境以促进细胞生长。
这通常意味着需要为细胞提供充足的营养和条件，例如适当的CO2浓度和恰当的温度。
在细胞转染过程完成后，需要进行逐步筛选和鉴定以评估转染的效果和细胞生长的状态。
总之，虽然原代细胞转染是一个复杂的过程，但结合我们了解的流程和注意事项进行，不仅能使转染过程容易和高效，同时也能提高转染的成功率，使我们更快速的满足实验的要求。
细胞转染步骤
细胞转染及其筛选1.传代：细胞于10cm盘消化后接种于6cm盘向6cm盘中加入3ml培养基,“米”字形摇晃。
当细胞生长到50％-70%时，根据细胞的生长速度可进行转染2.配置转染体系:(6cm盘)2。
4ug基因载体质粒；9ul转染试剂；200ul无血清培养基.混匀后室温静置10—15min孵育。
3.将6cm盘中的旧培养基弃去，换新的培养基3ml。
4.将配置好的转染体系加入到6cm盘中，混匀6-18h内换液。
5.12小时，换液后进行显微镜拍照观察。
6.再过6h加入G418，进行筛选(始终在6cm盘中进行）,使用G418浓度：600µg/mlG418的配置：取1gG418溶于1mlHEPES液中，加蒸馏水至10ml，过滤消毒4℃保存。
7.3-5天换液一次（换液不用PBS冲洗），此时仍要加G418，浓度不变,只到细胞呈单个细胞那种，当细胞死的过多时,可考虑药物浓度减半.筛选出稳定表达的cell。
8.挑克隆：1制备细胞悬液，细胞计数，用培养基稀释细胞到1个/10ul,在96孔板中加入培养基150ul/孔,在加入细胞悬液10ul/孔,待其逐渐增多后转入24孔板、6孔板、6cm 盘增殖。
2.伴随着细胞的生长,可能最后会变成细胞簇,故可以用黄枪头把细胞菌落挑出，放入24孔板里面。
9.单克隆鉴定:提取细胞RNA后，进行RT-PCR检测其表达量。
10.先做RT-PCR筛选一部分,在做western继续筛选一部分.所需物品:1。
转染试剂（Attractene Transfection Regent）；2.96孔板；3。
24孔板；4.6孔板；5.6cm盘；6。
G418 7. 1mlHEPES液。
细胞转染操作方法
细胞转染操作方法一、细胞转染简介细胞转染是指将外源性DNA、RNA、蛋白质等分子通过物理或化学手段导入到细胞内，从而改变细胞的基因表达或功能。
常见的细胞转染方法包括病毒载体介导转染、电穿孔法、钙磷共沉淀法和化学转染法等。
其中，化学转染法是最为常用的方法之一，因为它具有操作简便、成本低廉和适用于多种类型的细胞等优点。
二、实验前准备1. 细胞培养：选择适当的培养基和培养条件，使得细胞处于生长状态。
2. 转染试剂：选择合适的化学转染试剂，如Lipofectamine 2000、PolyJet等。
3. 质粒DNA：准备所需的质粒DNA，并进行纯化和测定浓度。
4. 培养皿：准备适当大小和形状的培养皿，以满足实验需要。
5. 显微镜：准备显微镜以观察细胞转染效果。
三、实验步骤1. 细胞接种：将需要转染的细胞接种到培养皿中，使其达到适当的生长状态。
2. 质粒DNA和化学转染试剂混合：根据试剂说明书的建议，将质粒DNA和化学转染试剂混合，形成转染复合物。
3. 转染复合物与细胞接触：将转染复合物滴加到培养皿中，让其与细胞接触。
4. 培养：将培养皿放入恰当的培养箱中，并按照所选细胞类型的要求进行培养。
5. 观察效果：经过适当时间后，使用显微镜观察细胞转染效果。
若需要，可以通过Western blot、PCR等方法进行进一步验证。
四、注意事项1. 选择适当的化学转染试剂，并按照说明书建议操作。
2. 转染前需要保证质粒DNA纯度和浓度足够，并进行必要的测定。
3. 细胞密度和培养时间应根据所选细胞类型进行调整。
4. 转染复合物与细胞接触时间不宜过长或过短，一般在4-6小时左右。
5. 培养过程中需要注意细胞状态的变化，并根据需要进行适当的处理。
五、实验结果解读通过细胞转染操作，可以实现外源性DNA、RNA、蛋白质等分子的导入，从而改变细胞的基因表达或功能。
实验结果可以通过Western blot、PCR等方法进行进一步验证。
细胞sirna转染操作流程
细胞sirna转染操作流程细胞siRNA转染是一种常见的实验操作，用于沉默特定基因的表达。
下面我将从多个角度全面地介绍细胞siRNA转染的操作流程。
1. 实验准备：a. 准备所需的细胞培养基、细胞培养皿、siRNA转染试剂（如Lipofectamine™ RNAiMAX等）、siRNA、目的基因的控制siRNA、PBS等。
b. 在转染前，需要将细胞培养至适当的密度，确保细胞处于良好的生长状态。
2. siRNA转染操作流程：a. 将需要转染的细胞计数并分配至培养皿中，使得在转染时细胞密度达到合适的水平。
b. 在一个离心管中混合适量的siRNA转染试剂和无血清培养基，轻轻振荡混合，并静置15分钟。
c. 将siRNA转染试剂混合物滴加到含有细胞的培养皿中，轻轻摇晃培养皿使转染试剂均匀分布。
d. 将细胞培养皿放回培养箱中，根据试剂的要求进行培养，通常在转染后的24-72小时内进行下一步实验。
3. 转染后处理：a. 根据实验需求，在转染后的适当时间点进行细胞的取样或者其他实验操作。
b. 如果需要进行长期实验或者观察，可以在转染后适当时间内更换培养基。
c. 对于不同的细胞系和siRNA转染试剂，最佳的转染条件可能会有所不同，因此需要根据实验要求进行优化。
总的来说，细胞siRNA转染是一个常用的实验技术，通过沉默特定基因的表达，可以帮助研究人员探索基因功能和细胞信号通路。
在进行操作时，需要严格按照试剂的说明书和实验要求进行操作，并且根据具体情况进行优化，以确保实验结果的准确性和可靠性。
希望这些信息能够帮助到你。
一文包揽细胞转染那些事儿（含操作视频）
一文包揽细胞转染那些事儿（含操作视频）作者：解螺旋·子非鱼如需转载请注明来源：解螺旋·医生科研助手导语细胞转染对初接触细胞实验的菜鸟而言，是一道难过的坎儿。
当科研汪们在这个坎儿上摔了无数次的跟头后，心里的阴影面积便被无限扩大，似乎只要碰到细胞转染就永无翻身之日。
然而现在就放弃该实验还为之过早。
正所谓授之以鱼不如授之以渔，同时也为了让大家成功跨过这道坎儿，小鱼这就把做细胞转染的技巧分享给大家，让你分分钟从菜鸟变成细胞转染达人！细胞转染是指将DNA或RNA导入真核细胞中，用于研究基因功能和蛋白表达分析等，可依据是否整合到细胞基因组，分为瞬时转染（24-96h）和稳定转染。
细胞转染方法类型及不同之处如下面两张图片所示，大家可以依据自己的实验需求来选择不同的实验方法。
其中，实验中应用最广泛的是脂质体介导的细胞转染。
细胞转染具体实验步骤的视频：一般影响转染效率的主要因素：细胞、血清、抗生素、核酸质量与数量、转染技术。
只要在这几个因素上多加注意，就可以避免掉进细胞转染效率低下的大坑。
细胞1. 选择传代次数较低并处于对数生长期的细胞进行转染。
对大多数细胞而言，均需要在转染当天或前一天铺板，第二天上午进行转染，48h后收集细胞进行功能检测。
对于原代细胞，可用促有丝分裂刺激物进行细胞活化。
2. 对于贴壁细胞，一般要求在转染前一日，用胰酶处理为单细胞悬液，重新接种于培养皿或瓶，最好在转染前4小时换一次新鲜培养液。
3. 支原体污染会严重降低细胞转染的效率，且支原体不会像细菌污染那么明显，因而转染前可用环丙沙星处理细胞以除去支原体。
4. 细胞转染时需要一定的细胞密度，以70-90%（贴壁细胞）或2×106-4×106细胞/ml（悬浮细胞）为宜。
核酸质量和数量1. 选择高纯度（OD260/280比值在1.8以上）且去除内毒素的DNA进行细胞转染。
如果DNA的纯度差，则其携带的少量的盐离子、蛋白、代谢物污染都会显著影响转染复合物的有效形成及转染的效率。
细胞转染 步骤
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细胞转染汉恒生物HANBIO
实验前一天铺板293T细胞准备转染；实验当天从细胞培养箱中取出前一天的细胞于显微镜下观察细胞密度，达到70%~80%的汇合率即可进行转染
①转染前，吸去细胞原有培养基，加入新鲜的完全培养基，轻轻八字混匀后放入培养箱中，待转染
以100mm培养皿为例，
②转染时取2个EP管，各加入500μl无血清培养基，然后一份加入常温溶解好的质粒，另一份加入本公司转染试剂lipofiter TM
③加好后轻轻振荡EP管，使之混匀，静置室温孵育5min
④5min后，将两个EP管中的液体混合，吹匀后静置室温孵育20min
⑤20min后，取出培养箱中的培养皿，加入EP管中混好的脂质体，8字摇晃后，放入培养箱培养6h
⑥6h后，取出培养箱中的培养皿，吸去旧的培养基，加入10ml完全培养基后放入培养箱，继续于37℃培养箱培养24h
⑦24h后取出培养皿，荧光显微镜观察转染效果。
细胞转染技术的使用教程
细胞转染技术的使用教程细胞转染技术是生物学研究和生物医学领域中一项重要的实验技术，它能够将外源基因或其他生物分子导入到目标细胞，从而改变细胞的性状和功能。
本篇文章将介绍细胞转染技术的基本原理、常用的转染方法以及技术操作的注意事项。
一、细胞转染技术的原理细胞转染技术通过物理、化学和生物学方法将外源基因或其他生物分子导入到目标细胞中。
常见的转染方式包括病毒介导转染、化学物质介导转染、电穿孔等。
病毒介导转染是利用病毒作为基因载体，通过病毒的复制和传播机制将外源基因导入目标细胞。
化学物质介导转染则是利用化学物质改变细胞膜的通透性，使外源基因进入细胞。
电穿孔则是利用高压电脉冲破坏细胞膜的完整性，使外源基因进入细胞。
二、常用的转染方法1. 病毒介导转染病毒介导转染是细胞转染中最常用的方法之一，常见的病毒载体包括腺病毒（Adenovirus）、慢病毒（Lentivirus）和腺相关病毒（Adeno-Associated Virus，AAV）等。
病毒载体能够高效地将外源基因导入目标细胞，并在细胞内稳定表达。
病毒介导转染具有转染效率高、表达时间长等优点，但也存在一些限制，如细胞感染范围狭窄、潜在的免疫反应等。
2. 化学物质介导转染化学物质介导转染常用的试剂有磷脂体（Liposome）和聚乙烯亚胺（Polyethyleneimine，PEI）等。
这些试剂能够与外源基因形成复合物，通过与细胞膜结合而进入细胞。
化学物质介导转染方法具有转染效率高、适用于多种细胞类型等优点，但也存在一些缺点，如细胞毒性、细胞内溶酪斑形成等。
3. 电穿孔电穿孔是一种利用高压电脉冲破坏细胞膜的完整性，使外源基因进入细胞的方法。
通过施加电场，电穿孔能够短暂地增加细胞膜的通透性，使外源基因能够进入细胞质。
电穿孔方法具有转染效率高、适用于多种细胞类型等优点，但操作相对复杂，需要专业的设备和技术支持。
三、技术操作的注意事项1. 细胞的选择和培养在进行细胞转染实验之前，需要选择适宜的细胞系进行培养。
转染操作步骤
转染操作步骤嘿，朋友们！今天咱来聊聊转染操作步骤这档子事儿。
转染啊，就好比是一场细胞的奇妙冒险！想象一下，你要把一些外来的东西，比如说核酸啦，送进细胞这个小小的“城堡”里。
这可不是一件容易的事儿，但别怕，跟着我一步一步来，保证能让你搞定它。
首先呢，得准备好你的“道具”。
细胞得养好啦，状态得杠杠的，就像要出征的战士一样精神。
还有那些转染试剂，可别小瞧它们，它们可是这场冒险的重要“伙伴”。
然后呢，就是关键的时刻啦！把核酸和转染试剂按照一定的比例混合起来，这就像是给它们牵红线，让它们“相亲相爱”。
搅拌均匀咯，可别马虎。
接下来，把这混合好的“魔法药水”慢慢滴加到细胞里。
嘿，这可得小心点，别跟细胞闹别扭。
滴加的时候，就像是给细胞送上一份神秘的礼物，得轻拿轻放。
之后呢，就是等待啦。
让细胞和这些外来的东西好好相处，给它们时间互相熟悉熟悉。
这时候你可别闲着，时不时去观察观察，看看有没有什么奇妙的变化。
过了一段时间，你就可以去看看成果啦！看看那些核酸是不是成功地住进了细胞这个“城堡”里。
要是一切顺利，哇塞，那可太棒啦！就好像你成功地完成了一项了不起的任务。
哎呀，这转染操作说起来简单，做起来可得细心再细心。
就像走钢丝一样，一步都不能错。
要是不小心出了岔子，那可就前功尽弃啦。
不过别担心，多练几次，你肯定能掌握其中的窍门。
转染这事儿啊，真的很神奇。
它能让我们把想要的东西送进细胞里，让细胞发生一些奇妙的变化。
这就像是给细胞施了魔法一样，是不是很有意思？所以啊，朋友们，大胆去尝试吧，别怕失败，失败是成功之母嘛！只要你有耐心，有细心，转染操作绝对难不倒你！加油哦！。
转染步骤
DNA转染步骤1 转染前一天接种细胞于6孔板，0.5-2×105每孔，2ml无抗生素培养基每孔，第二天转染时细胞达到90-95%每孔。
2 转染前半小时换Opti-MEM无血清培养基1.5ml。
3 A液：DNA 5ug 加入245ul Opti-MEM无血清培养基，轻轻混匀。
4 B液：脂质体使用前轻轻混匀，脂质体10ul 加入245ul Opti-MEM无血清培养基，轻轻混匀，室温静止5min。
5 B液加入A液，用枪轻轻混匀，室温静止20min。
6 将混合液均匀分散慢慢滴加到6孔板细胞中，边滴加变前后左右十字交叉摇晃。
7 孵箱37℃培养48h（转染时间待定）。
8 第二天看细胞状态来决定是否换液。
9 同时设立细胞对照组，空白转染组。
siRNA转染步骤1 转染前一天接种细胞于6孔板，2ml无抗生素培养基每孔，第二天转染时细胞达到30-50%每孔。
2 转染前半小时换Opti-MEM无血清培养基1.5ml。
3 A液：siRNA 5ul（共100pmol）加入245ul Opti-MEM无血清培养基，轻轻混匀。
（siRNA按照说明书稀释）4 B液：脂质体使用前轻轻混匀，脂质体10ul 加入245ul Opti-MEM无血清培养基，轻轻混匀，室温静止5min。
5 B液加入A液，用枪轻轻混匀，室温静止20min。
6 将混合液均匀分散慢慢滴加到6孔板细胞中，边滴加变前后左右十字交叉摇晃。
7 孵箱37℃培养48h（转染时间待定）。
8 第二天看细胞状态来决定是否换液。
9 同时设立细胞对照组，空白转染组。
实验室通常用细胞培养瓶与孔板实验室通常用细胞培养瓶与孔板。
siRNA转染方法
七 储存与安全
第4页 共4页
本品常温运输，储存于 4℃，有效期 12 个月。 本品使用安全，未发现任何生物、化学毒性。如不慎沾染，用清水冲洗即可。
八 其他相关试剂
EntransterTM-H：用于将 DNA 转染入 HEK 293(T)、Hela、CHO 细胞。 EntransterTM-D：用于将 DNA 高效低毒转染入动物细胞。 EntransterTM-in vivo：用于动物体内转染。 EnlightTM：高灵敏、低背景 ECL 发光试剂。
注意：在部分实验室，由于血清和培养条件等差异，转染后镜下培养基中可能出 现少量黑点状沉淀，为转染试剂和血清中蛋白结合产物，不影响转染结果和细胞 状态，可通过换液除去。
细胞培养容器
表 3 不同细胞培养容器转染推荐用量
表面积 (cm2)
表面积相对 于 24-well
比率
每孔
siRNA 用量
每孔 siRNA 每孔 EntransterTM 稀释液总体 EntransterTM-R -R 稀释液总
九 质量保证
北京英格恩生物科技有限公司对 EntransterTM-R 基因转染试剂的每批产品实行严 格质量检验，并进行转染验证，以确保产品质量。请用户使用前务必认真阅读本手册。
十 使用限制
本转染试剂仅限科研用途。
Copyright © Engreen Biosystem Co., Ltd (China). 2008-2010. All rights reserved. 版权所有 © 北京英格恩生物科技有限公司 2008-2010。保留全部权利。
1ml
6-well/35-mm 10
5
3.33μg 50μl
10μl
50μl
如何有效提高细胞转染实验效率（Entranster）
1．选择合适的转染试剂不同细胞系转染效率通常不同，但细胞系的选择通常是根据实验的需要，因此在转染实验前应根据实验要求和细胞特性选择适合的转染试剂。
每种转染试剂都会提供一些已经成功转染的细胞株列表和文献，通过这些资料可选择最适合实验设计的转染试剂。
当然，最适合的是高效、低毒、方便、廉价的转染试剂。
如英格恩的纳米材料的Entranster试剂。
2．保持最佳的细胞状态一般低的细胞代数（<50）能确保基因型不变。
最适合转染的细胞是经过几次传代后达到指数生长期的细胞，细胞生长旺盛，最容易转染。
细胞培养在实验室中保存数月和数年后会经历突变，总染色体重组或基因调控变化等而演化。
这会导致和转染相关的细胞行为的变化。
也就是说同一种系的细胞株，在各实验室不同培养条件下，其生物学性状发生不同程度的改变，导致其转染特性也发生变化。
因此，如果发现转染效率降低，可以试着转染新鲜培养的细胞以恢复最佳结果。
3．选择高效的转染方法不同转染试剂有不同的转染方法，但大多大同小异。
转染时应跟据具体转染试剂推荐的方法，但也要注意，因不同实验室培养的细胞性质不同，质粒定量差异，操作手法上的差异等，其转染效果可能不同，应根据实验室的具体条件来确定最佳转染条件。
4．确保所构建载体的质量。
转染载体的构建（病毒载体，质粒DNA，RNA，PCR产物，寡核苷酸等）也影响转染结果。
病毒载体对特定宿主细胞感染效率较高，但不同病毒载体有其特定的宿主，有的还要求特定的细胞周期，如逆转录病毒需侵染分裂期的宿主细胞，此外还需考虑一些安全问题（如基因污染）。
除载体构建外，载体的形态及大小对转染效率也有不同的影响，如前面提到的超螺旋及线性DNA对瞬时和稳定转染的影响。
如果基因产物对细胞有毒性作用，转染也很难进行，因此选择组成或可调控，强度合适的启动子也很重要，同时做空载体及其它基因的相同载体构建的转染正对照可排除毒性影响的干扰。
dsRNA细胞转染（Entranster）操作步骤
dsRNA细胞转染（Entranster）操作步骤
下面以Entranster-R4000试剂为例，说一下dsRNA转染步骤（24孔板）
1.提前1天细胞种植
贴壁细胞：提前一天将细胞种植在24孔板中，以转染时细胞汇合度（Confluence）在30％左右为宜，转染前全培养基总量为0.45ml。
悬浮细胞：采用对数生长期的细胞，数量为常规培养细胞数的1/3进行转染实验。
如某细胞常规培养的细胞数是6×105，那么就用2×105的细胞进行转染。
2.转染过程
⑴取1ug(50pmol)的dsRNA，加入一定量无血清稀释液，充分混匀，制成RNA稀
释液，终体积为25μl。
注意：无血清稀释液建议采用OPTI-MEM、无血清DMEM或1640。
⑵取1.5ul的Entranster TM-R4000，然后加入24ul无血清稀释液体，充分混匀，制成
Entranster TM-R4000稀释液，终体积为25μl。
室温静置5分钟。
⑶将Entranster TM-R4000稀释液和RNA稀释液充分混合（可用振荡器振荡或用加样
器吹吸10次以上）混合，室温静置15分钟。
转染复合物制备完成。
⑷将50μl转染复合物滴加到有0.45ml全培养基（可含10%血清和抗生素）的细胞
上，前后移动培养皿，混合均匀。
注意：对本试剂，采用含血清的全培养基有助于提升转染效率。
⑸转染后6小时观察细胞状态，如状态良好可不必更换培养基，继续培养24-96小
时得到结果。
动物体内转染（Entranster）答疑
动物体内转染（Entranster）答疑动物体内转染答疑----用RNA或DNA直接注射动物完成干扰和表达动物体内转染，简单地说，就是用RNA和DNA直接打动物完成干扰和表达。
再通俗地说，用合成的(RNA)或者提取的核酸(DNA)，就可以完成以前的动物转基因或者基因敲除的实验，无需再用病毒或者基因敲除动物。
实验周期可以缩短为几天，花费几千元即可进行实验。
动物体内转染技术的出现，让广大生物医学研究者，轻松进行动物的基因干扰、导入等操作。
尤其是临床医学工作者，可以在很少工作量较少经费的情况下，直接针对研究的疾病进行动物实验，发表高水平文章。
比如在英格恩客户已发表的文章中，有尾静脉注射DNA研究治疗病毒性心肌炎，有皮下肿瘤注射miRNA 研究治疗结肠癌，有脑室注射siRNA研究脑缺血机理，有皮肤涂抹siRNA治疗皮肤瘢痕等。
这些研究都非常有临床和现实意义。
由于动物体内转染技术应用的时间不长，对这种崭新的技术人们还不太了解。
此次受丁香园邀请，特开此动物体内转染相关实验技术答疑专帖。
对站友们提出的问题给予解答，希望能够和大家相互学习，共同进步。
任何与动物体内转染有关的问题（包括实验设计、产品、实验过程、结果分析、文献等问题），大家尽管提出，我们会尽力解答，也欢迎站友们参加讨论。
技术资料目录：1.体内转染试剂的原理和方法1).动物体内转染技术可以做什么？2).体内转染的原理3).体内转染的过程4).体内转染需要的实验条件5).体内转染适合进行怎样的实验6).体内转染可以在哪些组织器官进行7).应用体内转染试剂发表的部分文献8).动物体内转染和病毒感染的比较9).动物体内转染和基因敲除的比较2.体内转染实验的设计1).需要的材料2).需要的时间3).需要的费用4).常见的结果检测方法3.体内转染过程相关问题及解答1).体内转染试剂对动物有什么影响？2).注射后，试剂是如何在体内分布的，有靶向性吗？3).转染试剂和核酸需要使用多少？提问与解答：1、如何技术上解决（排除）RNAi的非特异性？是否需要复原实验（Rescue Experiment）。
细胞转染sop
细胞转染步骤细胞转染是将外源分子如DNA，RNA导入真核细胞的技术。
它已成为一种研究基因表达调控，基因突变分析，蛋白质生产的常规方法，其应用范围越来越广。
细胞转染可分为瞬时转染和稳定转染两大类：瞬时转染，外源基因进入受体细胞后，存在于游离载体上，不整合在细胞的染色体上。
此时，外源DNA仍然以附加的形式存在于细胞内，因此，mRNA或蛋白质产物必须在短时间（1-3天）内进行测定或分析，而且质粒的人工构想和拷贝数可能会导致特异性调控元件失活或具有特异功能。
其优点是快捷、简单，易于对结果进行分析，因此成为启动子功能分析的首选方法。
稳定转染，外源DNA整合到宿主细胞的染色体上。
由于外源基因被整合到细胞的染色体中，使得调控区能更精确的模拟正常功能，对随后的转录分析没有时间限制。
缺点是需要进行药物筛选和细胞扩增，因此操作难度大，需要的周期较长。
转染的常见方法有：（一）物理介导法：电击法、显微注射法、基因枪法（二）化学介导法：DEAE-葡聚糖法、磷酸钙法、脂质体法（三）病毒介导法：腺病毒法、逆转录病毒法现在对于很多普通细胞系，常用的是瞬时转染方法中的脂质体法。
下面介绍下细胞转染的具体步骤：1.转染前准备：转染前一天，取生长状况良好的细胞，经胰酶消化成单个细胞后，计数。
根据实验需要，铺合适细胞量在板中，使第二天转染时的细胞密度达到80-90%。
2、细胞转染（1）在做转染实验前一般给细胞更换新鲜的完全培养基，并置于培养箱中继续培养。
最好是加入加血清但不含抗生素的培养基。
（2）准备几个无菌的1.5ml EP管，并做好标记。
一支EP管上标记DNA（即质粒的名字），一支EP管上标记lipofectamine2000。
（3）分别在两支EP管加入50微升的opti-MEM。
（4）在标记质粒的EP管里加入质粒，另一支EP管里加入脂质体。
（5）分别轻轻混匀，室温静置5min。
（6）再将含有质粒的opti-MEM培养基加入到含有脂质体的opti-MEM的培养基中。
vivo（动物体内转染试剂）使用手册
vivo（动物体内转染试剂）使用手册Entranster TM-in vivo（动物体内转染试剂）使用手册Cat. No. 18668-11 Size:1ml常温运输，储存于4℃。
一产品介绍英格恩生物公司（Engreen Biosystem Co, Ltd.）是专业的转染试剂研发生产厂商。
Entranster TM是英格恩生物公司研发合成的纳米聚合物转染试剂，该试剂采用纳米技术合成，是最新一代非病毒转染试剂。
由于纳米技术的应用，Entranster TM-in vivo在细胞转染过程中，表现了卓越的低毒、高效的性能。
本品可用于转染DNA，也可以转染RNA(如siRNA、miRNA、mimic和inhibitor)，可用于如下研究：●基因治疗研究●RNA干扰研究●蛋白功能研究本品显著的特点是方法简单、快捷，价格低廉，对动物没有明显的炎性反应，对操作者安全。
二使用前注意一般情况下，核酸(ug)和Entranster TM-in vivo (ul)按照1:2的比例使用。
也可调整比例从1:1.5到1:4自行优化使用。
核酸的具体用量和注射用量须根据靶器官大小、动物大小、给药途径决定，具体可参考下表(核酸的用量换算成μg计算)。
表1 给药途径与核酸用量给药途径建议的核酸用量最大给药体积尾静脉50μg 200μl-400μl脑室 2.5-1μg 5μl成年小鼠腹膜100μg 0.6ml-1ml皮下肿瘤 10-50μg 100μl脑室 2-5μg 20μl成年大鼠静脉 150-300μg 1-1.5ml 一些器官比如皮下肿瘤的用量，也可以通过先确定最终的注射量，然后根据表2推算出核酸的用量和转染试剂的用量。
比如最终的注射量是100ul，那么核酸用量一般为12.5ug，Entranster TM-in vivo的用量为25ul。
表2 100ul 转染复合物的组成25μl 纯水25μl 核酸溶液12.5μg 核酸核酸稀释液25μl 的10%葡萄糖溶液25μl 转染试剂100μl 转染复合物转染试剂稀释液25μl 的10%葡萄糖溶液三操作步骤下面以50μg 的核酸与100μl 转染试剂，总注射体积400ul ，成年小鼠尾静脉注射为例说明。
转染步骤及经验
转染步骤及经验
1.根据要进行体细胞转染的实验的特殊要求，选择适当的载体质粒，以及目标细胞。
2.制备转染的DNA样本：根据质粒的大小和实验的要求，严格控制样本的DNA浓度，并确保其质量良好，无杂质；
3.准备转染液：将转染的DNA样本和必要的添加剂放入适量的
PBS/DMEM中混合，形成转染液；
4.细胞放入转染液中：把细胞放入适量转染液中，使细胞与转染液充分混合；
5.细胞转染：细胞转染的方法有优化的转染技术（如脉冲转染）和非优化转染技术（如胞浆转染），根据实验对转染效果的要求，选择合适的转染技术；
6.细胞休眠：细胞休眠后，为后续实验提供了理想的条件，简化接下来的操作。
7.细胞筛选：有些载体质粒将特定的标记物（如GFP）植入细胞内，接着，使用不同颜色的染料或者发光技术，筛选出转染后的细胞，这将有助于后续的实验。
8.检测转染效率：使用细胞膜染色，PCR，蛋白表达分析来检测转染效率，以证实转染是否成功。
9.细胞接种：转染细胞分离后，接种到HMVEC或其他细胞上，以复制体系，以便进一步的研究。
细胞转染是一个复杂的过程，需要仔细地操作。
转染步骤及经验
转染步骤及经验转染是在生物实验中将外源基因导入到细胞中的常用操作步骤之一。
本文将介绍转染的步骤及经验，并提供一些实用的技巧，旨在帮助读者顺利完成转染实验。
一、实验准备在进行转染实验前，必须做好充分的实验准备工作。
以下是一些关键准备步骤：1.1 细胞培养与处理- 培养适量的目标细胞，并确保其良好的生长状态。
- 处理细胞，使其处于适宜转染的状态。
例如，对于贴壁细胞，可以使用酶消化将细胞分散为单个细胞。
1.2 载体准备- 准备带有目标基因的载体。
这可以是质粒、病毒等。
- 使用合适的方法制备高质量的载体DNA或RNA。
1.3 转染试剂- 准备合适的转染试剂，如转染剂、离子可溶液等。
不同类型的细胞需要使用不同的试剂。
二、转染步骤转染步骤的具体操作根据不同的实验目的和细胞类型可能会有所不同。
以下是一般的转染步骤：2.1 细胞处理- 将要转染的细胞洗涤并收集到离心管中。
- 注意不要创建过高的转染细胞密度，以免影响转染效率。
2.2 与载体混合- 将载体与转染试剂混合，形成载体-试剂复合物。
根据实验需要可做不同浓度的复合物。
- 注意操作过程中避免产生气泡，以防止对细胞的损伤。
2.3 加入转染复合物- 缓慢地将转染复合物滴加到细胞中，确保复合物均匀分布在细胞表面。
- 转染时间和温度应根据细胞类型和所用试剂进行优化。
2.4 培养细胞- 按照细胞所需的培养条件将细胞孵育在恰当的培养基中。
- 在培养期间，根据实验设计进行后续处理，如观察表型、提取蛋白等。
三、经验分享3.1 优化试剂浓度- 不同的细胞系对试剂的敏感度不同，需要根据实验经验进行优化。
试验应根据需要尝试不同浓度的试剂。
3.2 选择合适的转染方法- 根据研究目的和所用试剂的特性，选择合适的转染方法。
常见的转染方法包括化学法、电穿孔法、病毒载体法等。
3.3 控制转染时间和温度- 不同细胞对转染复合物的吸收速度有差异，应该根据细胞类型和试剂特性确定合适的转染时间和温度。
DNA转染的方法(Entranster)研究IL-37b 基因
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（１． Ｍｅｄｉｃａｌ Ｃｏｌｌｅｇｅ ｏｆ Ｓｈｉｈｅｚｉ Ｕｎｉｖｅｒｓｉｔｙ， Ｓｈｉｈｅｚｉ ８３２０００， Ｃｈｉｎａ； ２． Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｃｌｉｎｉｃａｌ Ｌａｂｏｒａｔｏｒｙ， ｔｈｅ Ｆｉｒｓｔ Ａｆｆｉｌｉａｔｅｄ Ｈｏｓｐｉｔａｌ ｏｆ Ｓｈｉｈｅｚｉ Ｍｅｄｉｃａｌ Ｕｎｉｖｅｒｓｉｔｙ， Ｓｈｉｈｅｚｉ ８３２０００； ３． Ｌｉａｏｎｉｎｇ Ｍｅｄｉｃａｌ Ｃｏｌｌｅｇｅ， Ｊｉｎｚｈｏｕ １２１００１； ４． Ｋｅｙ Ｌａｂｏｒａｔｏｒｙ ｏｆ Ｓｐａｃｅ Ｍｅｄｉｃｉｎｅ Ｆｕｎｄａｍｅｎｔａｌｓ ａｎｄ Ａｐｐｌｉｃａｔｉｏｎ， Ｃｈｉｎａ Ａｓｔｒｏｎａｕｔ Ｒｅｓｅａｒｃｈ ａｎｄ Ｔｒａｉｎｉｎｇ Ｃｅｎｔｅｒ， Ｂｅｉｊｉｎｇ １０００９４）
RNA转染研究病理性色素沉着(entranster),转染效率高
A novel P53/POMC/G a s/SASH1autoregulatory feedback loopactivates mutated SASH1to cause pathologichyperpigmentationDing’an Zhou a,b *,Zhiyun Wei c ,Zhongshu Kuang a ,Huangchao Luo a ,Jiangshu Ma a ,Xing Zeng a ,Ke Wang a ,Beizhong Liu a ,Fang Gong a ,Jing Wang a ,Shanchuan Lei a ,Dongsheng Wang d ,Jiawei Zeng e ,Teng Wang b ,Yong He a ,Yongqiang Yuan a ,Hongying Dai a ,Lin He b,c ,Qinghe Xing b,*aDepartment of Laboratory Medicine,Yongchuan Hospital,Chongqing Medical University,Chongqing,ChinabChildren’s Hospital and Institutes of Biomedical Sciences,Fudan University,Shanghai,ChinacBio-X Institute,Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education),Shanghai Jiao Tong University,Shanghai,ChinadDepartment of Laboratory Medicine,The Affiliated Hospital of North Sichuan Medical College,Nanchong,ChinaeDujiangyan People’s Hospital,Cheng du,Sichuan,ChinaReceived:August 4,2016;Accepted:September 28,2016Abstractp53-Transcriptional-regulated proteins interact with a large number of other signal transduction pathways in the cell,and a number of positive and negative autoregulatory feedback loops act upon the p53response.P53directly controls the POMC/a -MSH productions induced by ultra-violet (UV)and is associated with UV-independent pathological pigmentation.When identifying the causative gene of dyschromatosis univer-salis hereditaria (DUH),we found three mutations encoding amino acid substitutions in the gene SAM and SH3domain containing 1(SASH1),and SASH1was associated with guanine nucleotide-binding protein subunit-alpha isoforms short (G a s).However,the pathological gene and pathological mechanism of DUH remain unknown for about 90years.We demonstrate that SASH1is physiologically induced by p53upon UV stimulation and SASH and p53is reciprocally induced at physiological and pathophysiological conditions.SASH1is regulated by a novel p53/POMC/a -MSH/G a s/SASH1cascade to mediate melanogenesis.A novel p53/POMC/G a s/SASH1autoregulatory positive feedback loop is regu-lated by SASH1mutations to induce pathological hyperpigmentation phenotype.Our study demonstrates that a novel p53/POMC/G a s/SASH1autoregulatory positive feedback loop is regulated by SASH1mutations to induce pathological hyperpigmentation phenotype.Keywords:SASH1 p53 DUH hyperpigmentationIntroductionThe skin pigmentation is originated from the synthesis of melanin in the melanocytes,followed by distribution and transport of pig-ment granules to neighbouring keratinocytes [1].Variations in the coding region of the melanocortin-1-receptor (MC1R)are impor-tant for tanning and pigmentation in human beings.MC1R is a G protein-coupled receptor (GPCR)that is preferentially expressed in epidermal melanocytes [2]and is activated by its ligand a -melano-cyte-stimulating hormone (a -MSH),a propigmentation hormone which is produced and secreted by both keratinocytes andmelanocytes in the skin following UV.a -melanocyte-stimulating hormone and other bioactive peptides are cleavage products of pro-opiomelanocortin (POMC),a multi-component precursor for a -MSH (melanotropic),ACTH (adrenocorticotropic)and the opioid peptide b -endorphin.Normal synthesis of a -MSH and ACTH is an important determinant of constitutive human pigmentation and the cutaneous response to UV [2].Within melanocytes,MC1R regulates the amount and type of pig-ment production and is a major determinant of skin phototype,*Correspondence to:Prof.Qinghe XING E-mail:qhxing@ Dr.Ding’an ZHOUE-mail:081023094@ª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley &Sons Ltd and Foundation for Cellular and Molecular Medicine.This is an open access article under the terms of the Creative Commons Attribution License,which permits use,distribution and reproduction in any medium,provided the original work is properly cited.doi:10.1111/jcmm.13022J.Cell.Mol.Med.Vol 21,No 4,2017pp.802-815sensitivity to UV radiation-induced damage and skin cancer risk[3]. Upon ligand binding,GPCRs impart a signal to heterotrimeric G proteins, which are composed of a-,b-and c-subunits,resulting in the detach-ment of the a-subunit from the G bc subunit of G proteins.G proteins of the G a s class directly catalyse the transformation of ATP to cAMP. cAMP is responsible for melanogenic actions of such ligands as a-MSH,including the activation of tyrosinase in melanin biosynthesis[4].The tumour-suppressor protein p53,a transcriptional factor,has been documented to directly activate transcription of numerous genes such as those that control cell-cycle,apoptosis and others.P53 directly mediates UV induction of POMC/MSH in skin and stimulates the POMC promoter in response to UV and is involved in UV-indepen-dent pathologic pigmentation and could mimic the tanning response [1].In the skin,p53function is critical for the retention of tissue integrity following UV irradiation[1].UV can exclusively induce dipyrimidine C to T substitutions that include CC to TT frameshift mutations in the p53gene,which were found in the skin of UV-irra-diated mice months before tumour development[5].In addition to the above activities,p53has been shown to be essential for the for-mation of‘sunburn cells’,which are a hallmark of sunburns[5].DUH is a clinically heterogeneous disorder that is characterized by generalized mottled pigmentation.DUH was initially described by Ichi-kawa and Hiraga in two generations of two families in1933[6].We discovered similar Chinese DUH pedigrees with dyschromatosis sym-metrica hereditaria(DSH)in2003with autosomal-dominant DUH[7] and diagnosed as DUH rather than DSH subsequently.However,the pathological gene and pathological mechanism of DUH have not been further characterized since itsfirst report in1933.SASH1was originally described as a candidate tumour-suppressor gene in the carcinomas of breast and colon and belongs to the previ-ously described novel family of putative adapter and scaffold proteins that transfer signals from the ligand to the receptor[8–10].Our previ-ousfindings indicate that SASH1binds to G a s,the downstream mole-cule of a-MSH/MC1R signalling cascade[11].Our previous study also showed that,in several DUH several affected individuals,hyperpig-mented macules were showed to become more pronounced after strong UV exposure especially in summer[7],but no further mechanism was identified the reasons of photosensitivity[12].The importance of expression of p53/POMC/a-MSH in UV-photopigmentation response and UV-independent hyperpigmentation has been elucidated[1].More-over,less observations were reported to demonstrate that the variations in SASH1gene are associated with hyperpigmentation and how these variations cause hyperpigmentation.Taken above,we hypothesise that a novel p53/POMC/a-MSH/G a s that SASH1is involved in,to mediate UV-photopigmentation response and pathological hyperpigmentation.Materials and methodsPCR,sequencing and mutation analysisTwo Chinese families from the Henan and Yunnan provinces of China and one American family with typical features of DUH were recruited for this study.Three pedigrees with DUH showed an autosomal-domi-nant inheritance pattern and were ascertained by experienced clinical dermatologists.The American family is a smaller pedigree,which could only provide three peripheral blood samples from affected indi-viduals for study.This research was approved by the ethical review committees from the appropriate institutions.Genotyping was per-formed,and the two-point LOD score was calculated as previously described[7].In total,50family members and500normal individuals (controls)participated in the study after providing informed consent. Samples of peripheral blood DNA were taken from all available family members.PCR and sequencing were performed as previously described [7].The sequencing was performed with an ABI BigDye Terminator Cycle Sequencing Kit(Applied Biosystems Inc,Foster City,CA,USA)on an ABI PRISM3130DNA Analyzer(Applied Biosystems),and data were analysed using sequence analysis software,version3.4.1(Applied Biosystems). Sequence data were compared with the SASH1reference sequence(Gen-Bank NM_015278.30)using Sequencher4.10.1(Gene Codes Corp,Ann Arbor,Michigan,USA).Nucleotide numbering reflects complementary DNA (cDNA)numbering,with+1corresponding to the A of the ATG translation initiation codon in the reference sequence[7].Construction of SASH1,G a s,POMC and p53 expression vectorsThe construction protocol of recombined vector of wt and mutant SASH1-PEGFP-C3and wt and mutant SASH1-PBABE-Flag-puro was mainly referred to our previous study[11].To construct HA-Pcna3.0-p53,myc-Pcdna3.0-POMC and GFP-G a s-Pegfp-C3vectors,PCRs of bacteria (obtained from Han jiahuai Lab,Xiamen University,Xiamen,China)contain-ing the vector of full-length CDS sequences of G a s,p53and POMC were performed with Phusion Hot Start High Fidelity Polymerase(New England Biolabs,Inc.,Ipswich,Massachusetts,USA)or GXL Polymerase(Takara, Shimogyo-ku,Kyoto,Japan),and the following primers were used:G a s primers50-ACGCGTCGACATGGGCTGCCTCGGGAAC-30(forward,Sal I site included)and50-CCGCTCGAG TTAGAGCAGCTCGTACTGACG-30(reverse, Xho I site included);p53primers50-CGCGGATCCGCCACCACCATGGAGG AGCCGCAGTCAGATCCTA-30(forward,BamH I site included)and50-CCG CTCGAGTCAGTCTGAGTCAGGCCCTTCTGT(reverse,Xho I site included); POMC primers50-CGCGGATCC ATGCCGAGATCGTGCTGC-30(forward, BamH I site included)and50-CCCAAGCTTT CACTCGCCCTTCTTGTA GGCGTTCTTGAT-30(reverse,Xho I site included).Mammalian expression vectors(Invitrogen,Carlsbad,California,USA)via the relative restriction sites were sequenced.Cell culture and transfectionA375cells,SK-MEL-28cells and HEK-293T cells were maintained as previously described[13].Normal human epithelial melanocytes (NHEMs,C-12402;PromoCell,Germany)were cultured in M2medium. A375,SK-MEL-28and HEK-293T cells were transfected using Lipofec-tamine2000(11668-027;Invitrogen)as previously described[13,14] or Entranster-D(18668-01;Engreen Biosystem Co.,Ltd,New Zealand) or polyethyleneimine(PEI)prepared by ourselves.The transfected A375 and SK-MEL-28cells were cultured in1.5l g/ml puromycin or2.0l g/ ml G418to select stable cell lines.HEK-293T cells were transiently transfected with wild-type and mutant SASH1-pEGFP-C3or co-trans-fected with wild-type SASH1-Pbabe-Flag-puro and G a s-Pegfp-C3ª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley&Sons Ltd and Foundation for Cellular and Molecular Medicine.803J.Cell.Mol.Med.Vol21,No4,2017vectors for immunoprecipitation experiments.NHEMs and HEK-293or HEK-293T cells were transiently transfected with Pcdna3.0-HA-p53, Pcdna3.0-myc-POMC,Pegfp-C3-G a s and wild-type SASH1-pEGFP-C3 according to pairwise combination to analyse the expression of exoge-nous p53,POMC,G a s and SASH1using PEI prepared by ourselves or PromoFectin(PK-CT-2000-MAC-1;PromoCell,Heidelberg,Germany).HEK-293T cells were transfected with G a s-GFP,HA-p53,myc-POMC and GFP-SASH1vector and subsequently silenced by G a s-and POMC-specific siRNAs that were synthesized by Shanghai GenePharma Co., Ltd(Shanghai,China)using Entranster TM-R Transfection Reagent (18668-06;Engreen Biosystem Co.,Ltd).The sense and antisense strands of each siRNA for G a s,POMC,GAPDH and the negative control are shown in Table S3.Pull-down assay and nano-flow LC-MS/MS and bioinformatic analysisThe protocols for the pull-down assay,nano-flow LC-MS/MS,database search and bioinformatic analysis for functional classification are mainly referred to our previous report[11]. Immunoprecipitation and immunoblottingHEK-293T or HEK-293transfected cells and NHEMs were gently washed in PBS three times and then lysed ing IP-Western blot lysis buffer(P0013;Beyond Time BioScience and Tech-nology company,Jiangshu,China)in the presence of a complete protease inhibitor cocktail,1l M sodium orthovanadate and1mM sodiumfluoride per10-cm dish on ice.Cell lysates were transferred into 1.5-ml microcentrifuge tubes.Extracts were centrifuged for 10min.at13400g.at4°C.Then,600l l of supernatants was pre-cleaned with20l l of Protein A/G PLUS-Agarose(sc-2003;Santa Cruz Biotechnology,Inc,California,USA)for1hr,immunoprecipitated using6l l of GFP-Tag(7G9)mouse mAb(M20004,Shanghai Abmart,Inc.,Shanghai,China)or6l l of DYKDDDDK-Flag-Tag mouse mAb(M20008;Shanghai Abmart,Inc.)or6l l of HA-Tag mouse mAb(SG4110-25;Shanghai Genomics,Shanghai,China)at4°C for 10hr and mixed with20l l of Protein A/G PLUS-Agarose(sc-2003, Santa Cruz Biotechnology,Inc.)at4°C for4hr and assayed using co-immunoprecipitation or immunoprecipitation.The immunoprecipi-tates were washed with PBS three times and subjected to SDS-PAGE and Western blotting.The primary antibodies used in the Western blot analysis were GFP-Tag mouse Ab(M20004,Shanghai Abmart,Inc.), Flag-tag mouse mAb(M20008;Shanghai Genomics),anti-G a s rabbit polyclonal Ab(G7X105877;Gene Tex,Inc.,Irvine,CA,USA),myc-tag mAb(SG411-30,Shanghai Genomics)and HA-tag mouse mAb(SG4110-25,Shanghai Genomics),SASH1Rabbit mAb(A302-265A-1,Bethyl Laboratories,Inc.,Montgomery,Texas,USA),DYKDDDDK-Flag-Tag mouse mAb(M20008;Shanghai Abmart,Inc.),TYRP1(TA99)mouse mAb(Ab3312;Abcam,Cambridge,UK),Rab27a mouse mAb (H0005873-M01;Abnova,Taipei City,Taiwan),melanoma gp100Rabbit mAb(ab137062;Abcam,Cambridge,UK),GAPDH mouse mAb(M20005; Shanghai Abmart,Inc.)and anti-b-tubulin mouse mAb(M20005M; Shanghai Abmart,Inc.).Immunoblotting was performed as previously described[15].Immunohistochemical and immunofluorescence staining,and melanin staining Immunohistochemical stainingWritten informed consent regarding tissue and data use for scientific purposes was obtained from all participating patients.Epithelial tissues from affected individuals with the Y551D SASH1mutation from pedi-gree family I werefixed in10%formalin at4°C for24hr and then embedded in paraffin.Paraffin sections(5l m)were incubated at56°C overnight and then deparaffinized and rehydrated using xylene and an ethanol gradient.The sections were incubated with the SASH1mono-clonal antibody(A302-265A-1;Bethyl Laboratories,Inc.),Rabbit Anti-ACTH(7-23)antibody(bs-004R;biosynthesis biotechnology Co.,Ltd, Beijing,China),Mitf polyclonal antibody(BS1550;Bioworld Technology, Inc,Louis Park,MN,USA),HMB45monoclonal antibody(sc59305; Santa Cruz Biotechnology,Inc.),TYRP1(TA99)mouse mAb(Ab3312; Abcam),Rab27a mouse mAb(H0005873-M01;Abnova)and p53mon-oclonal antibody(kit-0010-2;biosynthesis biotechnology Co.,Ltd)as well as horseradish peroxidase-linked anti-rabbit and antimouse univer-sal secondary antibodies or FITC.Finally,sections were counterstained with haematoxylin and photographed under the positive position micro-scope BX51.Immunofluorescence(IF)and confocal microscopyWild-type or mutant SASH1-A375stable cells were plated in6-well chamber slides and incubated at37°C for at least48hr.Indirect immunofluorescence analysis was performed on A375cells expressing wild-type and mutant SASH1(s)in6-well chamber slides to assess SASH1localization.IF was performed as described previously using the following antibodies:SASH1Rabbit mAb(A302-265A-1;Bethyl Labora-tories,Inc.)and DYKDDDDK-Flag mouse mAb(M20008;Shanghai Genomics)[11].Melanin stainingParaffin sections(5l m)from epithelial tissues were incubated in an 80°C baking oven for30min and then kept at room temperature for 15min.Melanin staining was performed according to the manufac-turer’s protocol(GMS80023.3;GENMED SCIENTIFICS INC.,Shanghai, China)and observed under a light microscope.Quantitative real-time RT-PCRThe total RNA from the different groups of SK-MEL-28cells was iso-lated using TRIzol Reagent(Invitrogen).Reverse transcription was car-ried out according to the manufacturer’s protocol for the PrimeScript TM RT Reagent Kit(DRR037A;Takara)or PrimeScript RT reagent using the gDNA Eraser Kit(DRR047A;Takara)for qRT-PCR.The sense and anti-sense primer sequences for SASH1,TYRP1,Pmel17,Rab27a,G a s, POMC and GAPDH are presented in Table S3.The PCR products were confirmed by agarose gel electrophoresis.Real-time PCR was per-formed using the Applied Biosystems7500System with SYBR Premix Ex Taq TM(DRR041A;Takara).The quantity of each mRNA was normal-ized to that of GAPDH mRNA.804ª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley&Sons Ltd and Foundation for Cellular and Molecular Medicine.UV exposureThe human foreskin tissues from a14-year-old boy were exposed for enough time under an UV phototherapy instrument(NBUVB SS-05; Sigma-Aldrich,St.Louis,Missouri,USA)to reach the expected UV intensity,thenfixed in10%formalin and embedded in paraffin for immunohistochemistry analyses.We conformed to the guidelines of the World Medical Assembly(Declaration of Helsinki)to acquire the human foreskin tissues.In the case of in vitro UV experiments which mainly referred to the protocol of our institute[16],HEK-293T cells and NHEMs transiently transfected with myc-POMC were cultured to approximately70–80% confluence in6-cm-diameter dishes and were irradiated with100mJ/ cm2UVC delivered via a HL-2000HybriLinker with a254-nm wave-length(Upvon)and followed by the indicated recovery time.Finally, cells were harvested to detect proteins’levels using immunoblot. Electrophoretic mobility shift assayThree probes binding with/without biotin,which targeted SASH1pro-moter,were synthesized.The sequence of probes was as follows:probe 1#50-GCCCAAGCTT TCACACTTGTTT-30,probe2#50-CCAAGACTTGCTA-GAAGGAACGAGTCG-30,probe3#50-CGTGGCCACCTAGACCCGAGGTG-30. Electrophoretic mobility shift assay was performed as described as the protocol provided with LightShiftâChemiluminescent EMSA Kit(20148; Thermo Scientific,Pierce Biotechnology,Rockford,USA). Statistical analysisThe data are presented as meanÆstandard error of the mean (S.E.M.)s.These data werefirst analysed using the homogeneity of variance test and followed by the change of variable test.Statistical significance was determined by a one-factor analysis of variance (ANOVA)with LSD correction on SPSS version16.0(IBM(International Business Machine))to generate the required P-values.Cartograms were plotted using GRAPHPAD PRISM5(GraphPad Software, Jolla,CA,USA)5.ResultsMutations in SASH1in DUH-affected individuals result in the up-regulation of SASH1in vitro and in vivoWe have located the gene that is responsible for DUH is local-ized to chromosome6q24.2-q25.2[7].The10.2-Mb region on chromosome6(6q24.2-q25.2)that isflanked by the markers D6S1703and D6S1708contained more than50candidate genes. We screened selected genes in this region for possible pathologi-cal mutations by directly sequencing the PCR products of exons that were amplified from genomic DNA of affected,unaffected and control individuals.We sequenced50candidate genes and found three heterozygous mutations encoding amino acid substi-tutions in SAM and SH3domain containing I(SASH1)in the probands in each of the two non-consanguineous Chinese DUH-affected families(families I and II)and in one non-consangui-neous American DUH-affected family(family III).SASH1point mutations were found in the three pedigrees.These mutations were as follows:a T?G substitution at nucleotide2126in exon 14in family I,a T?C substitution at nucleotide2019in exon 13in family II and a G?A substitution at position2000in exon 13in family III.These three nucleotide changes cause non-con-servative missense mutations in the SASH1gene,resulting in the following amino acid substitutions:Tyr to Asp at codon551 (TAC?GAC),designated as Y551D;Leu to Pro at codon515 (CTC?CCC),designated as L515P;and Glu to Lys at codon509 (GAA?AAA),designated as E509K(Fig.1A).These sequence changes were confirmed in all of the affected family members but were not observed in unaffected family members,correlating the presence of the mutations with the presence of the pheno-type.The mutations were not observed in any of the500normal controls or in any of the current databases,including the Hap-Map database.Therefore,these mutations are unlikely to be common single nucleotide polymorphisms(SNPs)[7].When SASH1mutants were stably expressed in A375cells,they significantly up-regulated SASH1(Fig.1B).Immunoblotting demon-strated that SASH1was up-regulated in A375cells stably express-ing either wild-type(WT-A375cells)or mutant SASH1(mutant-A375cells,including E509K-A375cells,L515P-A375cells and Y551D-A375cells),compared to the expression of endogenous SASH1in A375cells expressing the pBABE-puro empty vector (VECTOR-A375cells)or A375cells without any transfected vector (BLANK-A375cells)(Fig.1B).To verify the stability of SASH1pro-teins,HEK-293T cells stably expressing wild-type or mutant SASH1 were treated with20l g/ml of the protein synthesis inhibitor cyclo-heximide(CHX)for the indicated times to assess the half-life of SASH1.The protein levels of SASH1decreased in a time-course-dependent manner in response to CHX treatment.Wild-type SASH1 levels decreased with a half-life of approximately4hr.However,with CHX treatment for6hr or longer,CHX began to degrade mutant SASH1proteins.Therefore,the three mutant SASH1proteins were more stable than the wild-type,supporting the above observation that SASH1mutants are expressed at higher levels than the wild-type (Fig.S1A and B).Endogenous SASH1was an unstable protein with a half-life of approximately3hr(Fig.S1C).We characterized the subcellular localization of SASH1in A375cells and skin epithelial layers.The endogenous SASH1 protein in VECTOR-A375cells and the skin epithelial layers from normal controls demonstrated a homogeneous pattern of expres-sion(Fig.1C and Fig.S2-a4).However,in WT-A375cells and mutant-A375cells,activated SASH1(through either the overex-pression or mutation of SASH1)showed a pattern of heteroge-neous expression(Fig.S2-b4to Fig.S2-e4).The heterogeneous pattern of SASH1in vitro was also observed in vivo(Fig.1C).In addition,most of the SASH1-positive cells were melanocytes that were nucleic positive for Mitf,a melanocyte marker,andª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley&Sons Ltd and Foundation for Cellular and Molecular Medicine.805J.Cell.Mol.Med.Vol21,No4,2017Fig.1Mutations in SASH1increase SASH1expression in vitro and in vivo.(A)Mutation sites in the SASH1gene in three families with DUH.(B) Western blotting demonstrated the differential and increased expression of mutant SASH1proteins compared to that of wild-type SASH1in different A375cells.(C)HE staining,SASH1and Mitf immunohistochemical analysis of the epidermal tissues from the Y551D-mutation DUH-affected individ-uals and normal controls.Heterogeneous expression of the SASH1protein was observed in all of the epithelial layers in the epidermal tissues from the Y551D-mutation DUH-affected individuals as compared with that of normal controls(NC).Heterogeneous distribution of melanocytes was detected in the epithelial layers of DUH-affected individuals using the melanocyte marker Mitf as compared with that of normal controls.409magni-fication.Scale bar=20l m.Red arrows denote the representative positive cells of SASH1and Mitf.806ª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley&Sons Ltd and Foundation for Cellular and Molecular Medicine.demonstrated a heterogeneous distribution of melanocytes in the epithelial tissues of DUH-affected individuals as compared with those of unaffected individuals.Some cytoplasm-positive staining of Mitf is false positive (Fig.1C).The phenomenon thatmelanocytes or SASH1-positive epithelial cells localized not only to the basal layers but also to the suprabasal layers of the affected epidermal tissue is coincide with our previous conclusion that SASH1mutations promote melanocyte migration[11].Fig.2G a s interacts with SASH1and is a pivotal downstream of p53/POMC cascade.(A )The associations between GFP-SASH1and endogenous G a s were identified by immunoprecipitate-Western blot (IP-WB)analysis in HEK-293T cells.HEK-293T cells were transfected with the pEGFP-C3-SASH1vectors.At 24hr post-transfection,GFP-SASH1was immunoprecipitated (IP),and the associated GFP-SASH1was detected by Western blot analysis using an anti-GFP antibody.Different sizes of G a s bands were observed,at 28,46,68and 111kD,which may be caused by post-transla-tional modifications (PTMs).(B )GFP-G a s is associated with Flag-SASH1.HEK-293T cells were co-transfected with the pEGFP-C3-G a s and pBABE-puro-Flag-SASH1vectors.At 36hr post-transfection,Flag-SASH1was immunoprecipitated,and the associated GFP-G a s was detected by Western blot analysis using an anti-GFP antibody.(C )and (D )P53,POMC and SASH1is necessary for the activation of G a s.HEK-293cells and NHEMs were transfected with HA-p53,myc-POMC and GFP-SASH1,respectively,according to different manners of combination.After 36hr after transfection,two normal cells were lysed and subjected to immunoblotting with GAPDH as loading control.(E )Exogenous G a s is induced by p53.HA-p53and GFP-G a s were introduced into HEK-293cells.After 36hr after transfection,cells were lysed and subjected to immunoblotting.Exogenous G a s was activated by gradually increased amounts of exogenous p53(HA-p53).(F )Exogenous G a s is induced by SASH1.GFP-G a s and GFP-SASH1were introduced into HEK-293T cells.Exogenous G a s was induced by gradually increased doses of exogenous SASH1.(G )and (H )Exogenous p53(HA-p53)overexpression induces exogenous POMC(myc-POMC)expression in a dose-dependent manner in HEK-293T cells and NHEMs.Different dose of HA-p53vector and a certain amounts of myc-POMC vector were transfected into HEK-293T cell for expression.Exogenous POMC RNA levels were measured by quantitative RT-PCR and normalized to GAPDH.Results of RNA levels are expressed as the mean of the experiment carried out in triplicate Æthe S.D.The expression of HA-p53and myc –POMC was analysed by Western blot as GAPDH as loading control.ª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley &Sons Ltd and Foundation for Cellular and Molecular Medicine.807J.Cell.Mol.Med.Vol 21,No 4,2017SASH1is associated with G a s and induced by the canonical p53/POMC/G a s cascadeThe functional domains of SASH1(SAM and SH3)suggest that this protein plays a role in a signalling pathway as a signalling molecule adapter or as an associated scaffolding protein[8,9].Therefore,we performed a pull-down assay and a mass spectrometry analysis to investigate which signalling pathways are activated by SASH1.The pull-down assay and LC-MS/MS analysis demonstrated that SASH1 interacts with G a s and CALM,both of which are important in melano-genesis process(Table S1)in WT-A375cells.G a s is a key element of the classical signal transduction pathway linking receptor-ligand inter-actions with the activation of adenylyl cyclase and a variety of cellular responses[17].To investigate the associations between SASH1and G a s,HEK-293T cells were co-transfected with Flag-SASH1and GFP-G a s.Exogenous SASH1was immunoprecipitated with both exoge-nous G a s(GFP-G a s)and endogenous G a s.Exogenous SASH1 immunoprecipitates had different observed band sizes of G a s (Fig.2A and C).G a s mediates cAMP production in melanocytes which is stimu-lated by a-MSH and melanocortins[18],and our study here shows that G a s is associated with SASH1.Hence,we examine whether G a s is required for the induction of SASH1and how G a s mediates SASH1 expression,we introduced exogenous p53,POMC,G a s and SASH1 gene into HEK-293T and NHEMs to assess the effects of p53and POMC on G a s.Exogenous G a s was induced in the co-existence of exogenous p53and POMC(Fig.2C lane5and Fig.2D lane5),and both inducements of exogenous G a s and exogenous SASH1were observed in the co-existence of exogenous p53and POMC in two types of normal cells(Fig.2C lane6and Fig.2D lane6).Mean-while,in the presence of GFP-SASH1,GFP-G a s was also induced (Fig.2C lane4and Fig.2D lane4),which indicated that SASH1is necessary for the activation of GFP-G a s.And immunoblot showed that G a s was identified to be induced by exogenous p53and SASH1(Fig.2E and F).Our results also demonstrated that POMC was mediated by p53in HEK-293T and melanocytes were consis-tent with previous conclusions[1](Fig.2G and H).Conversely, endogenous SASH1and exogenous SASH1were induced by G a s (Fig.3A and B).To confirm the fact that POMC,p53and G a s are necessary for the inducement of SASH1,exogenous POMC,p53,G a s and SASH1were transfected into HEK-293T cells and followed by silence of G a s and POMC by two specific pairs of siRNA,respectively.As identified in HEK-293cells,knockdown of G a s gene directly induced signifi-cant reduction in SASH1(Fig.3C and D).Silencing of POMC resulted in the down-regulation of G a s and SASH1(Fig.3E and F).Taken above,it is believed that G a s serves as a pivotal downstream of p53/ POMC cascade and SASH1is regulated by a novel p53/POMC/G a s cascade.SASH1is physiologically induced by p53upon UV stimulationTo verify that SASH1is induced physiologically by p53,discarded normal human foreskin specimens were exposed to gradually increased dose of UV and stained for the histological analyses of p53, ACTH/POMC and SASH1.Immunohistochemical(IHC)analyses revealed p53is rapidly induced in basal layers at the0.5J/cm2dose of UV irradiation.The rapid induction of SASH1and POMC/ACTH at 1.0J/cm2dose of UV irradiation in melanocytes is followed by p53 up-regulation(Fig.4A).Previous reports had suggested that the up-regulation of POMC gene is induced at both protein and mRNA levels following UV irradiation of skin[19,20].Followed the previous descriptions[1],a100J/m2UVB dose was administered in this experiment.This dose is equivalent to the standard erythema dose (SED),which is commonly used as a measure of sunlight[21].So HEK-293T cells and NHEMs were transfected with exogenous POMC and followed by UV irradiation,both endogenous p53and SASH1pro-tein levels were assessed by immunoblot.UV markedly induced expression of exogenous POMC and endogenous SASH1by6hr,and p53induction was already maximal by3hr,which is consistent with its known stabilization by UV in NHEMs.At24hr,the levels of POMC, p53and SASH1protein were maximally induced by UV in NHEMs (Fig.4B).Similar inducement of exogenous POMC and endogenous p53and SASH1by UV irradiation was observed in HEK-293T cells (Fig.4C).Hence,we believe that not only POMC but also SASH1acts as a novel downstream partner which is responsive to the induction of p53by UV irradiation.Reciprocal induction between p53and SASH1is induced in normal cellsTo examine whether p53is required for the induction of SASH1, we introduced exogenous p53and POMC gene into HEK-293T and NHEMs to assess the induction of p53and POMC to SASH1. Exogenous SASH1was induced by p53in the presence of POMC (myc-POMC)in NHEMs and HEK-293T cells(Fig.S3).Exogenous SASH1was induced by increasing amounts of exogenous p53in two normal cells(Fig.5A and B).Conversely,exogenous p53was promoted by increasing amounts of exogenous SASH1(Fig.5CFig.3A novel p53/POMC/G a s/SASH1cascade regulates the expression of SASH1.(A)and(B)Endogenous or exogenous SASH1is induced by G a s.Gradually increasing amounts of exogenous G a s(GFP-G a s)and exogenous SASH1(GFP-SASH1)or only different doses of exogenous G a s were transfected into HEK-293T cells.The expression of endogenous or exogenous SASH1was analysed by immunoblotting along with GAPDH as loading control.(C)and(D)G a s is necessary for the inducement of SASH1.After transfection with GFP-G a s,myc-POMC and GFP-SASH1as well as increasing doses of HA-p53according to different combinations,two groups of HEK-293cells were subsequently introduced with two pairs of effective G a s siRNAs and negative control(NC)siRNA.Protein levels were detected by immunoblot.(E)and(F)POMC is necessary for the induce-ment of SASH1and G a s.After introduction into GFP-G a s,myc-POMC and GFP-SASH1as well as increasing dose of HA-p53according to different manner of combinations,two groups of HEK-293cells were subsequently silenced with two pairs of effective POMC siRNAs and NC siRNA.808ª2016The Authors.Journal of Cellular and Molecular Medicine published by John Wiley&Sons Ltd and Foundation for Cellular and Molecular Medicine.。
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下面以Entranster-R4000试剂为例，说一下dsRNA转染步骤（24孔板）
1.提前1天细胞种植
贴壁细胞：提前一天将细胞种植在24孔板中，以转染时细胞汇合度（Confluence）在30％左右为宜，转染前全培养基总量为0.45ml。
悬浮细胞：采用对数生长期的细胞，数量为常规培养细胞数的1/3进行转染实验。
如某细胞常规培养的细胞数是6×105，那么就用2×105的细胞进行转染。
2.转染过程
⑴取1ug(50pmol)的dsRNA，加入一定量无血清稀释液，充分混匀，制成RNA稀
释液，终体积为25μl。
注意：无血清稀释液建议采用OPTI-MEM、无血清DMEM或1640。
⑵取1.5ul的Entranster TM-R4000，然后加入24ul无血清稀释液体，充分混匀，制成
Entranster TM-R4000稀释液，终体积为25μl。
室温静置5分钟。
⑶将Entranster TM-R4000稀释液和RNA稀释液充分混合（可用振荡器振荡或用加样
器吹吸10次以上）混合，室温静置15分钟。
转染复合物制备完成。
⑷将50μl转染复合物滴加到有0.45ml全培养基（可含10%血清和抗生素）的细胞
上，前后移动培养皿，混合均匀。
注意：对本试剂，采用含血清的全培养基有助于提升转染效率。
⑸转染后6小时观察细胞状态，如状态良好可不必更换培养基，继续培养24-96小
时得到结果。