This plasmid was made based on PLKO shRNA backbone. You can design and clone your shRNA of interest just like you would for PLKO constructs. In brief, you would want to synthesize two annealing oligos according to GPP portal guides. For cloning, you would want to digest pAAVs1-shRNA plasmid by EcoRI and AgeI, and then clone the annealed oligos. What's different about this plasmid is that your shRNA of interest will be flanked by two homology arms that can target the construct to the AAVS1 locus. This will make one able to efficiently make stable cell lines without using a virus. Of course, you would want to co-transfect the plasmid with a CRISPR/gRNA or TALEN construct to make a double-strand break in the AAVS1 locus. These constructs are already available on Addgene (this one should work). The construct constitutively expresses puromycin resistant gene for selection.
2) pgRGFP (Plasmid #82695) This is a backbone that expresses the gRNA of interest and it also constitutively expresses GFP. This plasmid is made based on PX330, and cloning gRNAs follows the same principle (BpiI digestion of the backbone and cloning of annealed oligos).
3) pCRE-iRFP670 (Plasmid #82696) This plasmid constitutively expresses CRE recombinase and iRFP670 for cell sorting. It's a great plasmid for genome-editing situations where you want to flox out an insert flanked by lox-p sites. The iRFP670 is bright far-red bright fluorophore that is easily detectable on FACS or cell sorter in APC channel.
My paper went live on Cell Reports today. Aside from the joy that a scientist usually experience in such occasions, this publication coincided with two other occasions: 1) I attended Phill SHarp's talk at MIT, and he happened to give a very interesting talk on divergent and enhancer lncRNAs. 2) Today was October 4th, for all the stem cell biologists this is a reminder of OCT4, the infamous mother of all pluripotent cells...
The new results of our ongoing collaboration on the emerging field of epitranscriptomics is now live on Nature Methods. This paper describes a novel method to quantify levels of m6A methylation for specific transcripts and how this affects their alternative isoforms. Here's the link to the paper.
A while ago, I came across "Manuscript App", a software that, once fully developed, will help academics write their manuscripts and grants. The software is in developing stage and it is not released yet. I got lucky to be an early beta tester and I am now using it for a short manuscript that I am writing with my summer intern. Here are some great potentials of "Manuscript app":
1) It's simple: just by comparing its interface with other word editing softwares, you can obviously recognize and appreciate the simplicity.
2) Syncing: work on your paper at home, then go work on it at work (I don't know if there is going to be a cloud and web application or not).
3) There are academic templates for grants and papers.
4) There is possibility of citing as you write (I am assuming that you can import you library from other reference mangers such as mendeley)
5) You can export as submission ready manuscript! (This one is hard to resist!).
Summer is going downhill and this postdoc is happy! Yeap! I'm not your typical post-doc who would enjoy eating under the sun and talking about all things summery. After all, I live in Boston... so, I'm keeping calm and waiting for snow to come back!
Enough of prelude...
I finally got to make some updates to this website. Changes to my CV, research interest (believe me, this one is changing..!) and few other contents. More changes are on the way!
A while ago, I wrote about an extensive collaboration between our lab and four other labs at Harvard, MIT, UCLA and Stanford on a project about m6A RNA modification. Now the paper is available online (Cell Stem Cell) and will be in print soon. Although not being my primary research, the result of this paper is very interesting and it opens many doors for more investigations. I am excited to see how the scientific community will further develop this field in near future. Here is the link to the paper: m6A RNA Modification Controls Cell Fate Transition in Mammalian Embryonic Stem Cells
"Crunch time" is not an unfamiliar phrase for academics. With deadline for grant proposals, meetings registration and abstract submissions, internal deadlines for completing projects, we always have a deadline of some sort. As a postdoc, you are directly or indirectly involved in meeting many of the deadlines. Enough has been said about how to manage your time and be a multi-tasker in order to become a hero during the crunch times. What I have found as important is the necessity of keeping a healthy mind and body during these times. I don't know what your particular hobby is, but I highly recommend against compromising them just because you are on a crunch time. If cooking is what makes a good distraction for your mind, never settle for a pizza to secure more time for your academic work.
It has been a long time since I reported on my progress and things that I am currently doing. Here are some brief updates:
1) I really enjoyed the Keystone symposia on lncRNA. It was a great opportunity to interact with the people of the field. The Keystone symposia being very small and specific, I can describe the people that I met as my future collaborators, potential reviewers and editors of my scientific work...and who knows, maybe good friends! In fact those were all true!
2) We currently have a big publication in review. The paper brought together some six laboratories all across the east and west coast together. My contribution to the paper is not form my primary work on lncRNAs, but It relates to the system that I work with (i.e. embryonic stem cells). I will write more about this paper when it is in press.
3) I have more exciting new about my genome editing toolboxes. In the past few months I have had many constructive talks with the prominent people of the field and I have received many good feedbacks that make me believe that our genome editing toolbox will be a very efficient one for the community.
4) I am at the stage of investigating the role of my candidate lncRNAs in hES cells. There are some great early signs that make me optimistic about future!
5) Finally, spring is in Boston! It was a bitter and long winter, so I believe that we all deserve a long spring!
I had almost forgotten the pain of designing and printing a poster 24 hours before a conference! I am heading to Santa Fe for Keystone Symposium on long non-coding RNAs. This would be my first attendance at Keystone symposia, which is supposedly one of the very focused conference series. I am show casing one of the lncRNAs that I have been working on for the past year. This lncRNA is a divergent transcript from a protein coding gene. This lncRNA is particularly interesting to us as it seems to be relevant to the context of pluripotency and differentiation. I will be presenting some of our findings on characterization and functional analysis of this long non-coding RNA. More importantly, I will present some of the tools that I have developed, during my postdoc, for studying non-coding RNAs.
Science apart, I am excited to visit Santa Fe! It is going to be my second visit to New Mexico, but my first visit to Santa Fe! looking forward to it.