miRNA细胞转染Entranster步骤
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。
细胞转染的详细过程
细胞转染的详细过程1、准备工作如下：1)从pFastBacTM construct中纯化重组的bacmind DNA(500ng/μl溶于TE中)从相应的pFastBacTM construct对照中纯化bacmind DNA(500ng/μl溶于TE中)。
细胞培养在适合的培养基中。
细胞转染剂Cellfectin（4℃储存）无任何添加物（例如：FBS，抗生素等）的细胞培养基。
用于细胞培养的完全生长培养基（例如：Sf-900ⅡSFM TNMFH 或其他适合的培养基）。
2)在6孔板或是35毫米dish上，每孔培养9*105Sf9细胞，细胞培养于2ml含抗生素的生长培养基中。
3)细胞在27℃孵育至少一小时。
4)对于每一个转染的样品，准备bacmid DNA：与细胞转染剂在12*75mm消毒管中进行如下混合：用100μl无血清培养基稀释1μl纯化的bacmid DNA。
用100μl无血清培养基稀释6μl 细胞转染剂。
将bacmind DNA与细胞转染剂进行混合，动作要轻柔，混合物在室温下孵育45分钟。
5)当DNA与脂质体进行孵育时，移去细胞原有的培养基并用2ml无血清培养基洗一次，移去用来清洗的无血清培养基。
6)在每一个含有DNA与脂质体混合物的管子中加入0.8ml无血清培养基，轻柔混合，分别把DNA与脂质体混合物加入含有细胞的孔中。
7)细胞在27℃孵育5小时。
8)从细胞中移去DNA与脂质体混合物加入2ml完全生长培养基。
9)将细胞在27℃进行孵育，实验人员必须每日观察，记录转染细胞的生长状态，细胞在转染后48小时长势应该比较良好，但72小时后直至可以看到明显的病毒感染的细胞病变。
2、第一代代病毒的收集与保存1)当转染的细胞呈现出感染后期的形态时，收集每孔含有病毒的上清，转移到灭菌的15ml 压盖管中。
2)以500*g离心5分钟，从而移去上清中所含有的细胞及大的碎片。
3)将离心后的上清移到新的15ml压盖管中，用来保存第一代病毒，存放于4℃避光保存。
细胞转染操作方法
细胞转染操作方法转染，是将外源性基因导入细胞内的一种特地技术。
随着基因与蛋白功能讨论的深化，转染目前已成为试验室工作中常常涉及的基本方法。
转染大致可分为物理介导、化学介导和生物介导三类途径。
电穿孔法、显微注射和基因枪属于通过物理方法将基因导入细胞的范例；化学介导方法许多，如经典的磷酸钙共沉淀法、脂质体转染方法、和多种阳离子物质介导的技术；生物介导方法，有较为原始的原生质体转染，和现在比较多见的各种病毒介导的转染技术。
抱负细胞转染方法，应当具有转染效率高、细胞毒性小等优点。
病毒介导的转染技术，是目前转染效率最高的方法，同时具有细胞毒性很低的优势。
但是，病毒转染方法的预备程序简单，经常对细胞类型有很强的选择性，在一般试验室中很难普及。
其它物理和化学介导的转染方法，则各有其特点。
需要指出的一点，无论采纳哪种转染技术，要获得最优的转染结果，可能都需要对转染条件进行优化。
影响转染效率的因素许多，从细胞类型、细胞培育条件和细胞生长状态，到转染方法的操作细节，都需要考虑。
一、细胞传代1. 试验预备：200ul/1mlTip头各一盒(以上物品均需高压灭菌)，酒精棉球，废液缸，试管架，微量移液器，记号笔，培育皿，离心管。
2. 弃掉培育皿中的培育基，用1ml的PBS溶液洗涤两次。
3. 用Tip头加入1ml Trypsin液，消化1分钟(37℃，5%CO2 )。
用手轻拍培育瓶壁，观看到细胞完全从壁上脱落下来为止。
4. 加入1ml的含血清培育基终止反应。
5. 用Tip头多次吹吸，使细胞完全分散开。
6. 将培育液装入离心管中，1000rpm离心5min。
7. 用培育液重悬细胞，细胞计数后选择0.8X106个细胞加入一个35mm培育皿。
8. 将合适体积完全培育液加入离心管中，混匀细胞后轻轻加入培育皿中，使其匀称分布。
9. 将培育皿转入CO2培育箱中培育，其次天转染。
二、细胞转染1. 转染试剂的预备① 将400ul去核酸酶水加入管中，震荡10秒钟，溶解脂状物。
动物体内转染(Entranster)答疑
动物体转染答疑----用RNA或DNA直接注射动物完成干扰和表达动物体转染，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.体转染试剂的原理和方法1).动物体转染技术可以做什么？2).体转染的原理3).体转染的过程4).体转染需要的实验条件5).体转染适合进行怎样的实验6).体转染可以在哪些组织器官进行7).应用体转染试剂发表的部分文献8).动物体转染和病毒感染的比较9).动物体转染和基因敲除的比较2.体转染实验的设计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.体转染过程相关问题及解答1).体转染试剂对动物有什么影响？2).注射后，试剂是如何在体分布的，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）。
RNA转染试剂,高效率转染
Entranster TM-R4000（用于将RNA转染入动物细胞）使用手册一产品介绍本品推荐用于将siRNA、microRNA(miRNA)、mimic、inhibitor等小片断RNA转染入动物细胞（包括各种细胞系、原代细胞、悬浮细胞、昆虫细胞等）。
本品在多种细胞系的验证中均表现了很好的RNA转染效率，并有很低的细胞毒性，而较低的细胞毒性对RNAi实验尤其重要。
二重要提示1.由于本品细胞毒性很低，所以采用本品进行转染的细胞数量相对较少，这有助于提高转染效率。
2.采用本品进行转染，RNA用质量(ug)进行计量，对21nt双链的siRNA来说，1OD=3.0nmol=40ug。
3.siRNA等较短RNA请参照表1中siRNA用量，mRNA、shRNA等较长RNA请参照表1中mRNA用量。
4.如转染后需要用Trizol提取RNA，建议转染后6小时换液。
三实验过程（以24孔板siRNA转染为例）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0.67ug(50pmol)的siRNA，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1ul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！。
动物体内RNA转染操作步骤
动物体内RNA转染操作步骤下面以50μg的核酸与25μl转染试剂，总注射体积200μl，20g小鼠尾静脉注射为例说明。
局部注射不用稀释，直接根据需要把核酸和转染试剂混合即可使用。
1. 核酸的稀释。
将50μg核酸用适量无内毒素纯水稀释成1μg/μl，加入10%葡萄糖溶液(w/v)50μl，使葡萄糖终浓度为5%，终体积为100μl，充分混匀。
2. 转染试剂的稀释。
取25μl的Entranster TM-in vivo试剂用50μl的10%葡萄糖溶液稀释，并用纯水25μl补足，得到葡萄糖终浓度为5%，终体积为100μl液体，充分混匀。
3. 转染复合物形成。
立即将稀释后的转染试剂加入到稀释后的核酸溶液中，立即充分振荡混匀。
4. 室温静置15分钟。
配制好的转染复合物请即配即用，不宜长期存放。
5. 动物注射。
说明：1).尾静脉注射时请掌握注射技巧，一般选用远端1/3处静脉注射，如感觉到阻力和轻微隆起，请停止注射，重新寻找静脉进行操作，不要强力推注，否则容易将药液注射在尾部，导致尾部溃烂。
注射完成后移去针头，按压针孔10秒以上，防止药液流出。
2). 2.5mg/kg的给药剂量是起始给药剂量，如果动物可以耐受，可以按比例增加剂量，这样效果更好。
3).局部注射，尽可能多注射药液，有利于提高转染效果。
6. 基因表达检测。
一般来说，根据注射方法和靶器官的差异，12-48小时后基因表达效果较好。
7. 长期给药。
一次给药检测的最佳时间是注射后12-48小时。
如果需要维持长期效果，可以采用多次注射的方法，注射频度一般为每间隔2-3天一次，也可以根据实验情况适当延长至每7天一次。
详细描述细胞转染步骤
详细描述细胞转染步骤细胞转染是指将外源DNA或RNA引入细胞内，使其表达或干扰靶基因的过程。
该技术在基因工程、药物筛选和疾病研究中发挥着重要作用。
下面将详细介绍细胞转染的步骤。
1. 细胞培养准备在进行细胞转染之前，首先需要准备好细胞培养基和细胞。
常用的细胞包括哺乳动物细胞（如HEK293细胞、HeLa细胞等）和昆虫细胞（如Sf9细胞、S2细胞等）。
细胞应保持在良好的生长状态，通常需要在无菌条件下培养，并在适当的培养基中维持其生长。
2. DNA/RNA准备在进行细胞转染之前，需要准备好要转染的DNA或RNA。
DNA可以是质粒DNA、线性DNA或合成DNA，而RNA可以是mRNA或siRNA。
这些外源DNA/RNA应在实验室中进行合成或提取，并经过纯化、测序等步骤进行质检。
3. 转染试剂选择根据实验需要和细胞类型的特点，选择合适的转染试剂。
常用的转染试剂包括离子脂质体、阳离子聚合物和电穿孔等。
离子脂质体适用于多种细胞类型，而阳离子聚合物则更适用于特定细胞类型。
电穿孔则可用于绝大多数细胞类型。
4. 转染操作将细胞用适当的细胞培养基进行洗涤，以去除残留的培养基和细胞代谢物。
然后将细胞转移到新的培养基中，并使其适应新的培养条件。
接下来，将转染试剂与DNA/RNA混合，并在室温下孵育一段时间，以使其形成复合物。
然后将复合物加入到细胞培养基中，与细胞进行共培养。
5. 转染后处理转染后，细胞需要在适当的培养条件下继续培养，以使其表达或干扰靶基因。
在此期间，可以根据实验需要添加适当的选择抗生素或标记物，以筛选或鉴定转染细胞。
同时，还可以通过荧光显微镜观察转染效率，并进行定量分析。
6. 细胞检测和分析在转染后的一段时间内，可以通过PCR、RT-PCR、Western blot等方法，检测和分析靶基因的表达或干扰效果。
此外，还可以通过细胞增殖、凋亡、迁移等实验，研究转染对细胞生物学行为的影响。
7. 数据分析和结果解读根据实验结果，进行数据分析和结果解读。
细胞转染步骤
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500 a l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 C培养箱培养24h
⑦24h后取出培养皿，荧光显微镜观察转染效果。
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。
miRNA及其inhibitor转染方法
miRNA及其inhibitor转染方法1 对mimics、mimics control、inhibitor、inhibitor control进行稀释和分装（终浓度为100 nmol/μl）。
（1）Mimics加入250 μl DEPC处理的水；（2）Mimics control加入125 μl DEPC处理的水；（3）inhibitor加入250 μl DEPC处理的水；（4）inhibitor control加入125 μl DEPC处理的水；对以上进行分装：mimics 20 μl每管，mimics control 10 μl每管，inhibitor 20 μl每管，inhibitor control 10 μl每管。
分装后于－80 ℃保存。
2经过调整，细胞状态较好。
晚上铺细胞，4个60 mm培养皿，每皿7.0×105细胞。
做好标记，（1）为mimics（2）为mimics control（3）为inhibitor（4）为inhibitor control。
3上午八点对昨天晚上的细胞进行转染。
（1）a 用500 μl opti-MEM稀释20 μl mimics，作用5 minb 用500 μl opti-MEM稀释20 μl mimics control，作用5 minc 用500 μl opti-MEM稀释20 μl inhibitor，作用5 mind 用500 μl opti-MEM稀释20 μl inhibitor control，作用5 min（2）a 用500 μl opti-MEM稀释10 μl lip 2000，作用5 minb用500 μl opti-MEM稀释10 μl lip 2000，作用5 minc 用500 μl opti-MEM稀释10 μl lip 2000，作用5 mind 用500 μl opti-MEM稀释10 μl lip 2000，作用5 min（3）分别将（1）、（2）对应的a、b、c、d混合，轻轻吹打均匀后室温作用20 min。
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.。
质粒转化、细胞转染步骤
质粒转化、细胞转染步骤
转化：将质粒DNA或以其为载体构建的重组DNA导入细菌体内使之获得新的遗传特性。
转染（transfection）:将含有目的gene的载体转到真核cell内
转导：指以噬菌体为载体，在细菌之间转移DNA的过程。
有时也指在真核cell之间通过逆转录病毒转移和获得cellDNA的过程。
1.质粒转染（transfection）
6-well-plate每well60-70%时transfection
接种时2.5×105个/ml/well
质粒DNA转染液优化培养基opti-MEM
步骤：2.5ug质粒DNA+200ulopti-MEM+6ul转染液混匀室温静置15min 待转染cell换优化培养基opti-MEM1.3ml，切记要轻轻加培养基，勿冲起cell
把transfectioncomplex(200ul)轻轻均匀滴入cell培养基中，十字形摇晃plate,混匀
37℃，CO2培养箱培养
2.
转化：外源DNA(质粒作为载体)导入受体细胞（大肠杆菌感受态cell）步骤：
50ul感受态cell（一管）加入12ul质粒DNA→轻柔混合
↓
冰浴30min
↓
42℃热激90s
↓
迅速冰浴2min
↓
向管中加入培养基，混匀后37℃震荡培养（﹤225rpm）1h,使细菌恢复正常生长状态
↓
涂板正培1h后，倒培过夜。
细胞转染流程
细胞转染流程细胞转染就像是给细胞送一份特殊的“快递”，这个“快递”里装着我们想要细胞接受的东西，比如说新的基因之类的。
那咱们就开始这细胞转染之旅吧。
在做细胞转染之前，你得先把细胞照顾好。
细胞就像娇嫩的小宝贝，要给它们一个舒适的“家”，这个“家”就是培养皿啦。
得把细胞在合适的培养基里养着，温度要刚刚好，就像人住在温度适宜的房子里一样舒服。
要是温度不合适，细胞就会闹脾气，就像人在过热或者过冷的环境里会不舒服一样。
而且培养基里的营养成分得充足，就好比给孩子的饭菜得营养全面，这样细胞才能长得壮壮的，有精力来接受我们要给它的“快递”。
接下来就是准备转染试剂和要转染的核酸啦。
这转染试剂就像是专门的快递员，它的任务就是把核酸这个“包裹”送到细胞里面去。
核酸呢，可以是DNA，也可以是RNA，这就看你想让细胞做什么了。
你得按照说明书，精确地量取转染试剂和核酸的量。
这就好比做菜的时候，盐放多了或者放少了都不行，得恰到好处。
要是转染试剂或者核酸的量不对，那这个“快递”可能就送不好，细胞可能就接收不到正确的“包裹”。
然后就是把转染试剂和核酸混合在一起。
这时候就像是快递员把包裹打包好一样，要轻柔地操作。
不能太粗暴，不然可能会把“包裹”弄破或者弄乱了。
混合好之后，还得让它们静静地待一会儿，就像给快递员一点时间整理一下包裹，确保一切都准备妥当。
再把这个混合好的东西加到细胞培养皿里。
这就像是把打包好的快递送到细胞的“家门口”了。
细胞看到这个“快递”，就会想办法把它“拿”进去。
这个过程有点像细胞在门口迎接快递员，然后把包裹拿进自己的家里。
不过细胞拿这个“包裹”进家可不容易，有时候可能会失败，就像快递员有时候会送错地址一样。
在细胞接收这个“快递”的过程中，我们得在旁边静静地看着。
就像等着孩子拆礼物一样，充满期待又有点小紧张。
我们得观察细胞的状态，看看它们有没有什么不良反应。
如果细胞开始变得无精打采，或者形态变得奇怪，那就可能是转染出问题了。
microRNA过表达载体具体步骤及说明
microRNA过表达载体具体步骤及说明microRNA 过表达载体具体使用说明及步骤MicroRNA（miRNA）是一类真核生物内源性的小分子单链RNA，通常长为18~25nt，能够通过与靶mRNA 特异性的碱基配对结合，引起靶序列降解或抑制其翻译，从而对基因进行转录后的表达调控（如右图所示）。
GenePharma MicroRNA 载体利用CMV 启动子可以在众多哺乳动物细胞中快速、高效、持续表达pre-miRNA，经过Drosha（RNase Ⅲ）作用形成small hairpin pre-miRNAs。
在使用载体法针对某一MicroRNA 进行过表达研究过程中，通常会遇到如下几个问题：实验对照组的确立、细胞转染条件的确定、基因表达效率的检测。
1、实验对照组的确立在一个完善的MicroRNA 表达实验设计中，必须考虑设立正确合理的实验对照组。
通常，这些对照组包括载体阴性对照组、转染试剂对照组。
载体阴性对照可以有两种，一种是采用通用的阴性对照组，在本试剂盒中包括了该对照所需的载体（microRNA），可以表达与目的基因序列无同源性的microRNA 片段；另一种是将目的microRNA 的序列打乱后重新组合所得的阴性对照（scrambled）。
对照组的设立对于MicroRNA 表达研究是很有必要的，您可以利用载体对照来确认microRNA 实验中转染、RNA提取和基因表达检测方法的可靠性。
2、细胞转染条件的确定使用MicroRNA 载体转染细胞时，为了选择合适的转染方法和确定转染效率，通常采用报告基因来检测DNA 的导入情况。
最常用的报告基因是绿色荧光蛋白。
3、MicroRNA 表达的检测通常用两大类方法来检测MicroRNA 表达效率，一类方法是直接检测MicroRNA 的变化水平，常用的方法如northern 杂交、芯片（microarrays）以及核糖核酸酶保护分析，但这些传统的方法都需要用到标记探针与纯化的RNA 进行杂交，由于成熟的miRNAs 及其前体共享一段共同的靶序列，而且目前的这些方法有无法通过大小将其分开，因此很难专一识别成熟的MicroRNA，导致较高的背景信号。
细胞转染步骤
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.G4187. 1mlHEPES液-精品-。
miRNA的生物合成过程
MicroRNA(miRNA) 是近几年在真核生物中发现的一类具有调控功能的非编码RNA，它们主要参与基因转录后水平的调控。
miRNA 的调控功能是十分重要的，近来发现它们在果蝇的细胞增殖、死亡及脂肪代谢,线虫极性的形成，哺乳动物的造血干细胞的分化等过程中起了重要的调控作用。
在植物中，miRNA 还参与了叶子与花的发育过程。
动物(尤其是人) miRNA 的生物合成过程已经初步得到了诠释。
首先，miRNA 基因的初级转录产物（pri—miRNA) 在细胞核中被 RNase Ⅲ Drosha 切割成为前体 miRNA (pre—miRNA）。
在最初的剪切后，pre-miRNA 在转运蛋白 exportin—5 的作用下由核内转到胞质中，然后由另一种 RNase Ⅲ Dicer 进一步切割产生成熟的 miRNA.这些成熟的 miRNA 与其他蛋白质一起组成 RISC (RNA-induced silencing complex）复合体，从而引起靶 mRNA 的降解或者翻译抑制。
1 miRNA 基因的转录与转录初产物的剪接大多数 miRNA 基因与蛋白质基因距离比较远，它们可能有自己的启动子可以进行独立的转录。
但是也有相当多的 miRNA 基因，人有1/4的 miRNA 基因是位于蛋白质基因的内含子当中的，通常 miRNA 基因与内含子的转录方向是一致的，这就说明这些基因大多是与寄主蛋白基因共转录的，然后再从这些蛋白质基因的内含子中剪切出来。
并且同一类 miRNA 基因在不同生物体中的寄主基因往往是保守的同一类蛋白质基因.正是由于 miRNA 与其寄主蛋白基因是共表达的,而使它们的联系在进化过程中也保守地保存下来.还有一些 miRNA 基因是成簇分布在染色体上，它们通过一个共同的启动子转录成为多顺反子.虽然这种形式在线虫和人的 miRNA 基因中比较少见，但是果蝇的 miRNA 基因中一半是成簇的。
而且这些成簇的 miRNA 基因经常是彼此相关的.例如人的mir-15a-mir-16 基因簇是位于13号染色体上的一个抑癌基因中，而这一位置在 B 细胞与 T 细胞白血病中均产生了结构的畸变。
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下面以Entranster-R4000试剂为例，说一下miRNA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0.67ug(50pmol)的miRNA，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1ul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。