How do you make shRNA-expressing viruses for function screening?

Allele Weekly Blog: http://blog.allelebiotech.com/2011/11/how-do-you-make-shrna-expressing-viruses-for-function-screening/

Most people use standard cloning procedures when trying to insert shRNA templates into lentiviral vectors, i.e. anneal a pair of long oligos with sticky ends and ligate the dsDNA into a linearized plasmid with compatible overhangs. However, since typical lentiviral vector plasmids have terminal repeats and are relatively large, when ligated to hairpin sequence-containing shRNA templates, recombination often occurs inside bacteria that results in smaller plasmids. This problem is common for cloning shRNA or other unstable DNA pieces into viral vectors. This cloning issue is further compounded by the fact that it is difficult to sequence any shRNA template region because the hairpin may block the progress of the DNA polymerase used in sequencing, sometimes requiring several repeats under different sequencing conditions, incurring high costs charged by sequencing service providers.

To deal with these aspects of the cloning difficulties, particularly for the purpose of increasing cloning efficiency RNAi-based screening, we compared three different strategies

First, we built a smaller shRNA cloning vector to clone and sequence shRNA templates prior to transferring to lentiviral vectors. This smaller vector does not have a severe recombination problem and is easier to sequence in the hairpin-containing region. After an initial round of cloning with this new vector, we further improved it by inserting an XbaI and a NheI site between the BamHI and SpeI insertion sites, so that any plasmid preparations can be screened for recombinants by a simple XbaI or NheI digest before sequencing. After cloning into this intermediate vector, the shRNA expression cassette can be transferred into the lentivirus vectors with some flanking viral sequences so that the insert size will be around 1kb.

Second, we developed a novel DNA preparation procedure after realizing that DNA damage during miniprep of vector plasmids and gel purification of vector fragment increased recombination of these constructs, which were already less stable than usual due to hairpin structures. This procedure of DNA preparation avoids UV or guanidium exposure, which can cause nicks on double-stranded DNA and facilitate recombination. This new procedure relies on purifying DNA through surface-binding to regular reaction tubes treated with a proprietary reagent (SurfaceBind Purification). The process simply requires adding a proprietary, guanidium-free binding buffer to the DNA, which has been processed in a specially coated tube (eppendorf or thin-wall PCR tube), and purifying directly in the same tube. Vectors prepared this way indeed provide more colony counts and a higher percentage of correct constructs as shown by our test runs. The procedure also requires less time and the purified DNA can be dissolved in volumes as small as a few microliters.

Third, to enable truly high throughput shRNA screening (i.e. looking for effective RNAi reagents), we further tested and adapted a ligationless cloning protocol that can be handled by a liquid handler almost entirely. In order to increase throughput, we designed a drastically different procedure that could bypass ligation and sequencing altogether before functional tests. Briefly, DNA molecules that would provide enhanced recombination were created by one round of PCR, purified directly in the surface bind PCR reaction tubes (any template DNA would be removed with DpnI enzyme that cuts non-PCR DNA), pooled, and transformed in bacteria directly. DNA plasmids from transformed bacteria can be used for lentivirus packaging, bypassing sequencing at the initial screening stage, and choose single colonies for sequencing only after a shRNA sequence shows promise in functional assays. This is based on the fact that such cloning rarely has any background colonies, and that among all oligos (if using the correct grade of oligos from validated suppliers) inserted this way, a good portion encodes the correct sequence.

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Development of Cell Lines from iPSCs for Bioassays

The reprogramming of differentiated somatic cells to pluripotency holds great promise for drug discovery and developmental biology. Using immortalized cell lines for drug screening assays has its limitations, such as questionable relevance; and the use of primary cells is often hindered by supply difficulties. Thanks to pioneering work by the Yamanaka, Thompson, and other groups, the feasibility of creating iPSCs has generated an opportunity to provide cell lines with stem cell properties in a virtually unlimited supply [1, 2]. These cells can be derived into different cell types for specific assays, even with patient- or genotype-specific background. Technologies are being developed to produce re-differentiated cells of a number of lineages.

Take cardiomyocytes as an example. There are a number of conventional methods for inducing stem cells into cardiomyocytes: through embryoid body (EB) formation, co-culturing with visceral endoderm-like cell line (END-2), and monolayer caridomyocyte differentiation with defined growth medium and protein factors [3]. A recent publication showed that using appropriate concentrations of BMP4 and activin-A in BSA-containing medium cardiomyocytes might be achieved from iPSCs or ESCs in about 6 days [4].

Transdifferentiation, or direct reprogramming, by introducing a group of 3 cardiomyocyte-specific factors, investigators could directly program cardiac or dermal fibroblasts into cardiomyocyte-like cells [5]. Although much refinement and characterization of these directly reprogrammed cardiomyocyte-like cells, termed iCMs, will be needed before the process can become widely used, this work raised the possibility of quicker and perhaps more efficient ways of generating cells for assays. Similar transdifferentiation has resulted in induced neuron (iN) cells, also by introducing 3 tissue-specific transcription factors [6]. Therefore, it seems that by using defined combinations of tissue-specific transcription factors it is possible to generate cells of different tissue types. It is also possible that by using different, developmental stage-specific transcription activator sets, transdifferentiation can be conducted in a stepwise way and make sure cells at each step is pure. This strategy may be particularly attractive if its efficiency can be improved by the techniques developed for iPSC creation. After all, reprogramming to pluripotency and transdifferentiation to different tissue types must share certain mechanistic steps in their respective processes.

In addition, it has been reported that by briefly overexpressing the Yamanaka iPS factors and controlling growth conditions, mouse fibroblasts could be transdifferentiated up to 40% in 18 days without reversing back to pluripotency [7]. It would be interesting to see if by transient expression of iPS factors via mRNA then switching to cardiomyocyte-specific transcription factors, we can increase the efficiency for direct reprogramming. Use of chromatin-modifying chemicals that were already shown to directly reverse and alter cell fates might also be used to assist direct reprogramming. We believe that a systematic approach for studying these reprogramming aspects should benefit the iPS fields.

1. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76.
2. Yu, J., et al., Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007. 318(5858): p. 1917-20.
3. Vidarsson, H., J. Hyllner, and P. Sartipy, Differentiation of human embryonic stem cells to cardiomyocytes for in vitro and in vivo applications. Stem Cell Rev, 2010. 6(1): p. 108-20.
4. Elliott, D.A., et al., NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat Methods, 2011.
5. Ieda, M., et al., Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell, 2010. 142(3): p. 375-86.
6. Pang, Z.P., et al., Induction of human neuronal cells by defined transcription factors. Nature, 2011. 476(7359): p. 220-3.
7. Efe, J.A., et al., Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat Cell Biol, 2011. 13(3): p. 215-22.

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李承鹏：一只叫萨克斯风的破鞋

我小时候在新疆，特别喜欢看抓破鞋。因为相比于其他各种类型的坏人，搞破鞋的貌似长得好看些，也不像其他坏人被抓斗时尽说些很艰深的事情，破鞋说的通俗易懂。那时哈密有个露天的“小河沟电影院”，河水从天山化解而下，清凉蜿蜒，两岸稀拉长着些胡杨，破鞋们就从电影院出发，脖子上挂着破鞋，成双成对沿着河岸被游行，边走边交待怎么搞上的破鞋、如何接头、如何亲嘴……剩下的就不许讲了，但仅仅这样，已让我觉得很是有趣。



那时对破鞋的定义不仅是奸夫淫妇，在野地里搞对象也算搞破鞋，因为搞对象就该在屋子里搞，野外搞，当然就是搞破鞋。有个姓安的小伙总是被抓，因为不仅喜欢在野外搞破鞋，还要吹着萨克斯风。虽然当时在新疆，用萨克斯风进行汇报演出不算搞破鞋，可在野外对着女人吹萨克斯风就是十足的搞破鞋。跟其他人不一样，交待到最后，姓安的常被工宣队的要求来一段萨克斯风，他会面带微笑，吹上一段，很好听。让我从小就觉得萨克斯风就等于搞破鞋，而搞破鞋其实是件挺美好的事情。



我只是一直不明白，为什么给领导汇报演出时吹萨克斯风不算搞破鞋，搞对象时吹萨克斯风就算搞破鞋。这个问题，我听那个姓安的小伙也问过，工宣队大概表达了这样的意思：萨克斯风是外国乐器，要是吹锁呐问题就小些（注：当时还没上演红高梁），总之领导听什么都没问题，因为领导更有社会主义道德。



前段时间，国家广电总局决定限制各卫视娱乐节目，我觉得“限娱”没有问题，问题在于为什么只限制19：30-22：00的娱乐节目，不限制19：00-19：30的那档娱乐节目。那是一档看的人没当真、念的人没当真、写的人没当真、下命令的人更不当真，可大家集体假装很当真的样子且一当真就是几十年……的王牌娱乐节目。可现在的状况跟小时候很相像，工宣队与广电总局的逻辑也很一样：屋子里就是搞对象，野外就是搞破鞋；给领导汇报演出时吹萨克斯风不算搞破鞋，给对象吹萨克斯风就算搞破鞋……地面频道的不算娱乐，上了星就是娱乐，19：00-19：30不算娱乐，19：30-22：00就是低俗娱乐。



所以我国的事情一点没变，表面看在限娱，实际上还是在抓破鞋。



我常幻想，多年之后，天安门广场矗立起一座娱乐博物馆，盛况空前地记录着以下的抓破鞋：禁《流星花园》，限穿越剧，限网络音乐，限谍战剧，禁同性恋题材，声讨郭德纲，禁《蜗居》……这个国家太多的破鞋。我不解为什么要怕人民搞破鞋，其实让他们搞破鞋，就没精力搞破坏。竟听说八成群众纷纷支持限娱，表示“是该提升一下道德了”，哪天我一定要会会该名永远叫“八成”的群众，问他是不是“八戒”表亲。又听说文化部为避免暴力提升道德，决定推出“绿色游戏”，我好奇绿色游戏里的悟空是否不可拿金箍棒，只可拿祥云火炬……大家知道最近我国又很爱谈道德了，因为佛山两岁女孩被反复辗压十八路人却漠然置之，让长期在19：00-19：30里群HI的礼仪之邦很难堪。总得找出原因，却不能说是官德倒退让民德倒退，所以找来找去，终于在司机、路人、家长之外找到娱乐这个大破鞋。



抓娱乐这大破鞋符合这里一惯的政治逻辑。早年的《海瑞罢官》掀起文革，前两年中国音协声讨过低俗音乐，有个老同志痛心疾首：我很担心自己的孙子孙女，他们现在十多岁，很容易灵魂就受到网络低俗音乐的污染，很担心等他们成为社会中坚力量时这些恶劣影响还在。可并没见有谁受低俗音乐污染，因为这么轻易就污染，它就不是音乐，是原子弹。



把道德下降归罪于娱乐，这可太娱乐了；说娱乐败坏了道德，这本身就不道德。港台的娱乐就低俗，可没有七十码、地沟油和见死不救，人家买东西好好排队，保钓却也冲到最前头。至于领导说的高雅艺术以提升道德，我对这个可真是很怀疑。我还记得《辛德勒名单》有个情节：屠城的那个晚上，犹太人纷纷躲藏在楼梯间、墙体夹层。纳粹军就用听诊器去听墙体里有没有呼吸声。有个犹太人不小心碰到了钢琴，士兵们发疯般冲上楼去一通扫射，从而掀开了第二次屠杀的序幕。可在机关枪声、惨叫声中，长夜里忽然响起一阵悦耳的钢琴声，很优秀的琴声，流畅而激昂，有一种巴赫式的宗教宁静。两个士兵被琴声吸引，竟在门边讨论，一个说：是巴赫。另一个说：不，是莫扎特……我一直以为这是视死如归的犹太艺术家临终的演奏，可画面摇起，一个表情肃穆的纳粹军官，一个高雅艺术的爱好者。



纳粹军队可谓二战时期音乐素养最高的一支军队，希特勒和戈培尔都曾强力在军队推行高雅艺术。希特勒本人是瓦格纳的粉丝，德国空军轰炸伦敦前大多要听贝多芬《第三交响曲》，奥斯威辛集中营司令官克拉麦杀人时甚至要听舒曼的梦幻曲……可见高雅艺术提升道德是个伪概念，艺术欣赏力跟杀不杀人并没关系。否则以后监狱里不安狱警，安装一水儿的高保真黑胶唱机，罪犯也不越狱了。大街上要碰到绑匪，直接播出《众神的黄昏》，一听感动得化了：哈里路亚，不能杀人了，去唱诗班吧。



当然要提升道德，可不要用抓破鞋的方法去提升道德，也可以抓破鞋，可不要一边抓破鞋一边自己在搞最大一只破鞋。希特勒、戈培尔当初就用抓破鞋手段摒弃一切低俗艺术，甚至一度禁止电台播放爵士乐，因为爵士乐来自美国，这多么低俗。后来虽然允许在舞厅里演奏爵士乐，但已是只能用小提琴和大提琴演奏“洁本”了。想像一下，用小提琴和大提琴演奏的爵士乐，就跟中国小脚老太太跳芭蕾一样古怪了，可希特勒认为，这样的艺术改造才能让帝国的意志更统一、更强大、更能忘记痛苦。



所以现在还呼吁“人民有低俗的权利”的朋友就很不上道了，此时我真切地政治敏感到这次祖国真是要推动限娱——道德——文化的一体化强国工程。表面上是在限娱乐，其实在抓破鞋，表面在提升道德，其实在统一思想，又不好意思给没头脑的刁民明说，绕了好大一个圈子，你看，我们很早就不方便谈政治了，后来也不好谈历史了，谈地理其实也是敏感瓷，现在连风月都开始不许谈了，所以只好谈谈道德。限娱乐是为了抓破鞋、抓破鞋为了促道德、促道德必然结果是，建成一个正确的文化体系……



是该社会主义文艺复兴的时候了，像美国那么没道德的国家都能成文化大国，有道德的我们更是前程远大。虽然我们没有一个好大学，没有一部好电影，没有一个好作家，没有一个好博物馆，没有一档好电视节目，没有一个真实历史……但必须指出，我们有论语心得，有建党伟业，有孔子学院，有大爱无疆，有19：00-19：30，还有西门庆故居。虽然我们报刊杂志不太说真话，但印刷品数量是全球第一。虽然我们出版审查是严了一点，但实在不行，还可以出手抄本。虽然我们有个别无德贪官，但贪污几千万的十大品牌市长李启红“还是有很多好的品质，骨子里无比热爱党”。虽然我们的舆论监督遇到些问题，可监督舆论从来不是问题，你看前面我那篇文章，虽然只有一个标点符号，却能有三十多万点击率，这才叫传媒大国、文化强国，这才叫软实力，名副其实。



最后一个故事，是文章写到这里时发现Richard Overy介绍的：“上世纪三十年代早期，苏联视爵士乐为一种文化颠覆，跳爵士舞，也作为堕落的资产阶级生活方式。可是低俗堕落的资产阶级生活方式实在诱惑太大了，官方不得不让步，成立国营爵士乐团，但只允许演奏旋律柔和的舞厅曲目，或是改编自俄罗斯民歌的音乐。一九四五年以降，爵士乐因为冷战头号敌人美帝国主义，更是罪加一等。到了一九四九年，苏联萨克斯风的生产与销售皆为非法”。



让我们最后一次谈谈风月吧，原来老大哥早就抓获了一只叫萨克斯风的破鞋。一只叫萨克斯风的破鞋，一个叫李启红的道德，一个只剩下标点的文化。