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。

Creating ground-state human iPSCs

AlleleBlog

Murine pluripotent stem cells can exist in two distinct states, blastocyst-derived LIF-dependent embryonic stem cells (ESCs) and epiblast-derived bFGF-dependent stem cells (EpiSCs). Murine ESCs and similar iPSC lines are more of the “ground-state” in terms of developmental status, as reflected by the lack of X chromosome inactivation in female cells and their abilities to pass as single cells. Human iPSCs, like human ES cells, are more similar to mouse EpiSCs. Unfortunately these human pluripotent stem cells are difficult to genetically manipulate, e.g. knockin or knockout. They also grow slowly, with doubling time averaging 36 hours. In order to create ground-state human iPSCs, several approaches have been tested, including reprogramming iPSC-derived fibroblasts, continuously expressing 5 iPS factors (Oct4, Sox2, Nanog, c-Myc, and Klf4), or using chemicals to inhibit chromatin modifying enzyme HDAC. While these approaches succeeded to certain degrees, the resulting cell lines seem to have some limitations, such as limited passage numbers.

Retinoic acid (RA) signaling is involved in many aspects of embryonic development. RA receptor (RAR), together with one of its heterodimerization partners, steroid hormone receptor Lrh-1, was recently found to be able to synergize with the 4 common iPS factors (Oct4, Sox2, Klf4, and c-Myc) to induce mouse and human fibroblasts into ground-state iPSCs. The pluripotent cells created by the so-called F6 factor combination show no X chromosome inactivation if from female origin, can fully activate the endogenous Oct4 promoter, express Rex1 (which is specific to mouse ESCs, not EpiSCs), and grow with a 16 hour doubling time. All these mouse ESC-like features were achieved without detectable expression of the exogenous factors once iPSC colonies formed, indicating transient F6 expression is capable of effectively initiating endogenous stem cell factors. Remarkably, these stem cells can maintain their undifferentiated status in mouse ESC medium for 50 passages or more. This work, published this month in Proceedings of National Academy of Science USA [1], provided the stem cell research and application field with a very desirable choice of human stem cells.

As opposed to ~16 days with F4, it appears that the time required to induce adult fibroblasts into pluripotent stem cells is as short as 4 days if F6 factors are introduced on a murine stem cell virus (MSCV) vector with an integrated piggyback transposon. As the authors noted in their discussion, the speed-up benefit should be particularly advantageous for transient transfection approaches such as mRNA reprogramming. The bottom line from this paper and the engineered factor papers (see the previous AlleleBlog article under “iPS and other Stem Cells”) is that iPSC reprogramming is only going to get faster, which means that hopefully in the near future creating iPSCs will become a routine experiment as easy as a simple transfection.

Wang, W., J. Yang, et al. (2011). “Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1.” Proc Natl Acad Sci U S A.

New Products of the week: ARCA, modified cap analog for in vitro transcription of mRNA.

Promotion of the week: Friday special this week, 15% off all iPS viral particle products if using code “ViraliPS” when ordering online at allelebiotech.com, by email, or fax.

Solving the world’s problems with new biotechnology

The ability to isolate, create, synthesize, or artificially evolve living organisms towards desirable phenotypes may be increasingly important for solving many of the problems the world is facing. Such problems may include creating renewable energy using biowaste, finding biocontrol products that kill food-spoiling fungi “organically”, or assaying pathogens in the field using synthetic biological detection systems. With the arrival of synthetic biology, “it is possible to design and assemble chromosomes, genes and gene pathways, and even whole genomes”, according to the J. Craig Venter Institute. That is, if you know which genes or gene pathways you would need to put into the synthetic genome that would lead to the desired traits. So far, most published synthetic biology work involves bringing in transcription factors from a non-host source to set up an artificial network like circadian oscillators, showing that it can be done and it is interesting.

Through the process of evolution biological systems aptly self-engineer favorable traits in order to survive, but these changes require millions of years to manifest. However, there are quicker adaptations to environmental cues, such as developing antibiotic resistance, which can be achieved through a small number of mutations in hundreds or even dozens of generations. The question is how to harness this kind of adaptation for new strains that can be used as products with defined purposes? As a first requirement, you must have an assay for identifying the wanted mutants or method for augmenting their subpopulation, which is not necessarily easy and normally takes some clever designs to establish. Since evolutionary success in nature results from continuous “rounds” of gene mutagenesis, expression and selection, an evolution in the lab should ideally proceed with continuity. Previously, each round of mutation and selection takes a few days to complete. Recently, Esvelt et al. in David Liu’s lab at Harvard demonstrated one way of doing in vitro continuous evolution, by creating a lagoon of mixed E. coli and phages. By continuous dilution of the phage population through outflow, those phages that remain in the pool with properties that help them propagate in the host bacteria will have a better chance to regenerate and accumulate mutations towards the design of the assay [1].

AlleleBlog: http://blog.allelebiotech.com/2011/10/solving-the-world%e2%80%99s-problems-with-new-biotechnology/

Another aspect of natural evolution is that it occurs in a heterogeneous environment separated into niches of subpopulations with uneven stress levels. Although most evolutions with human intervention were conducted in a homologous population under the same stress and selection, a spatially complex environment may speed up evolution. This may not be easy to imagine, but if a mutant acquires some level of resistance to its environmental stress level and has a chance to move to join a population under higher stress, its relative fitness will likely increase. In addition, in a smaller population in the niche under higher stress, the mutant with marginally beneficial properties acquired under lower pressure can take over more quickly. This was demonstrated by Zhang et al. who showed that with a gradient of antibiotics applied to an array of microwells interconnected through tiny channels, new resistant strains can evolve in less than a day. Without the gradient, or separate the interconnected niches into discrete wells, no resistant populations could be obtained [2].

With more understandings like these and equipped with large scale gene synthesis, chromosome assembly, and deep sequencing technologies, we should see increasing numbers of human-made organisms serving special needs for food, health, energy, and the environment. Synthetic biology or artificial evolution won’t solve all the world’s problems, but if applied effectively and diligently, they can certainly help with many critical aspects as the technology “coevolves” with the environment.

[1] Kevin M. Esvelt, Jacob C. Carlson, & David R. Liu. “A system for the continuous directed evolution of biomolecules” Nature 499, 2011.
Qiucen Zhang, Guillaume Lambert, David Liao, Hyunsung Kim, Kristelle Robin, Chih-kuan Tung, Nader Pourmand, Robert H. Austin. “Acceleration of Emergence of Bacterial Antibiotic Resistance in Connected Microenvironments” Science 333, 2011.

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Promotion of the week: Friday special this week, buy 2 GFP-Trap get 1 free. Email the code “2+1GFPTrap” after placing your order of 2 GFP-Trap beads (0.25ml or 0.5ml scales only).

Originally at AlleleBlog at blog.allelebiotech.com

Storm, by 2011 Nobel Prize in Literature winner, Tomas Transtromer.

“突然，漫游者在此遇上年迈
高大的橡树――像一头石化的
长着巨角的麋鹿，面对九月的大海
那墨绿的城堡

北方的风暴。正是楸树的果子
成熟的季节。在黑暗中醒着
能听见橡树上空的星宿
在厩中跺脚”

——托马斯·特郎斯特罗姆《风暴》；李笠译《特郎斯特罗姆诗全集》

By 2011 Nobel Prize in Literature winner, Tomas Transtromer.

Fusion of the Transcription Domain to iPS Factors Radically Enhances Reprogramming

AlleleBlog:

Induced pluripotent stem cells (iPSCs) can be achieved through introduction of a small group of stem cell specific transcription factors. Ever since this was first demonstrated by Takahashi and Yamanaka, there have been relentless efforts for improving the efficiency of this generally inefficient process. There is also a general opinion that iPSCs are different from each other and from embryonic stem cells (ESCs) in various aspects, depending on the method of the induction. As a result, another focus of the reprogramming field has been to find ways for creating iPSCs that are as close to ESCs as possible. One of the parameters for defining stem cell status is their epigenetic characters; epigenetic changes have been demonstrated to occur during reprogramming of subsequent differentiation.

In fact, it seems that reprogramming can be largely described as a process composed of chromatin remodeling and specific transcription activation. Strong transcription activators are known to effectively recruit multiple chromatin remodeling complexes when exerting their functions. A good example is MyoD, a master transcription factor for skeletal myogenesis that can “single-handedly” switch (transdifferentiate) the fate of differentiated cells. Hirai et al. speculated that since MyoD is such a strong transcription factor, it may be able to increase chromatin accessibility to iPS factors if fused together. When transduced on retroviral vectors, Oct-TAD (Transcription Activation Domain) of MyoD, in combination with Sox2 and Klf4, increased the number of iPSC colonies by 40-fold. Additionally, these iPSCs appeared to quickly adopt stem cell gene expression profiles, days faster than when traditional Oct4, Sox2, c-Myc, and Klf4 were used; and sometimes the levels of pluripotency genes even exceeds those seen in ESCs. Amazingly, when using the fusion assisted method some colonies are formed without the help of feeder cells, a requirement of ESCs grown in similar medium. Does this mean that these iPSCs can even be more “stem-like” than embryonic stem cells?

Like MyoD, VP16, also widely known for its strong transcription activation domain, when fused to iPS factors, was shown to exhibit a similar stimulation effect on reprogramming. Although the details of the fusion arrangements and specificity appear to differ between MyoD and VP16, the fact that two research groups could achieve similar results using comparable strategies provides a good argument that other labs should at least consider this method when creating mouse or human iPSCs. Previously in our blog we have discussed using iPS factor mRNAs, a method originally developed by Warren et al., for substantially shortening the time required for reprogramming and making it more robust across cell types and media conditions. If the new TAD-fusion factors are used also in the mRNA format, then the protocol might be further shortened and simplified. If successful, this non-integrating approach could become a dominant method in the field, even making competitive non-integrating method such as Sendai and plasmid-based miRNA irrelevant.

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