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Hi,
No way to know yet actually since we have done this once just to check if there is a reduction in gfp which we observed both by facs and western. We are currently sequencing the products to rule out Rnai if any. Will let you know if that's confirmed
Best
Debo
Dr. Debojyoti Chakraborty
Room 129, CSIR-Institute of Genomics and Integrative Biology
Mathura Road,
New Delhi - 110025
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That's very good news Debo.Need some clarification tho. Did you use the Addgene plasmid or you design the construct w/ gblocks. Did you use the same protocol as it was mentioned on the paper? Can you share some pcr/blot pictures? Thank you for sharing.
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Hi Haijun,
Our transfection rates are pretty high, reaching 70-80% and NgAgo reducing plasmid expressed gfp is pretty reproducible. We have controls including non Phosphorylated gDNAs which do not give a reduction in GFP so what we see cannot be explained by transfection related issues. We are doing more tests to look at all possibilities and would be able to comment once we are done.
Best
Debo
Dear Xiaobing,
I would guess that too. So far controls have been scrambled and non Phosphorylated gDNAs. Haven't finished looking for indels or sequencing yet, guess that would be the most telling experiment..
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Yes, but which one is optimized for mammals? as everyone who got it from addgene has issues and you reported success and got it from miaolingbio it seems the one sent to addgene is not optimized for mammals.
so again I ask why no documentation supporting that? why not say for the addgene plasmid this is not codon optimized for mammals. but why even deposit that when most people interested are working with mammals, and they already have worked with human cells from the paper so they presumably could have sent addgene the mammalian optimized plasmid.
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Protein sequence alignment shows NLS-NgAgo-pcDNA3.1 (commercial) and nls-NgAgo-GK (addgene)
is almost identical to the NgAgo in GeneBank (ANC90309.1), except the following differences:
I don’t think codon optimization is that important. Although the commercial plasmid might express better than addgene one, still no Indel has been reported by Debo’s lab.
So why do we see a GFP knockdown? One possibility is that the NgAgo in cells might inhibitor transcription through binding to the target gene but it doesn't make any DSB.
It is quite disappointing, but I do believe that NgAgo system can be further improved. We certainly need more information from the author.
Best,
Xia
The band shift, distortion, and intensity raise some concern. But the images are probably heavily compressed during publication and quite poor in quality. High quality raw files are needed to address the concerns.
Please have a look at these pictures from Han's paper.
Concerning your "one-guide-faithful" claim, what indications do you have that a NgAgo protein doesn't swap guides in vivo? Based on your experimental setup (fig2 and SupFig1), I understand that when NgAgo is incubated at 37C it doesn't bind the guide (using lack of activity as a proxy). So, why the incubation (at 37C) of NC pre-loaded NgAgo, extracted from cells, with the FW guide tells you that there is no guide swapping? I would like to see if swapping happens at 55C. Even though this is not a "physiological" temperature and this incubation would reduce the activity of NgAgo (to nickase at best), this experiment would reveal if there is an intrinsic ability of guide swapping.
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