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研究者称成功重复韩氏 NgAgo 方法

已有 10647 次阅读 2016-7-4 12:54 |个人分类:反民科|系统分类:海外观察

我虽然是外行看热闹,但字还是认识的。大家可以看看。该研究者称已经成功运用韩氏方法,目前正在进行基因测序确认。从讨论看,大家仍然在探索之中,而且并非完全重复韩论文,而是有所变化。

We have used this system and it works. We have followed the same protocol as mentioned in the paper and were able to confirm knockout both on FACS as well as western.

该研究者 Dr.Debojyoti Chakraborty 履历好像比方舟子强一个量级或以上。参见《对韩春雨论文的质疑不应在暗处》。


Dr.Debojyoti Chakraborty

Post Doctoral Researcher [December 2013 – November 2015]

Max Planck Insitute of Molecular Cell Biology and Genetics & Technical University, Dresden,Germany

PhD in Molecular Biology, 2014 [Magna Cum Laude]

Thesis title: Systematic dissection of long noncoding RNAs involved in the regulation of embryonicstem cell pluripotency

German Stem Cell Network Travel Award, Frankfurt, Germany [2015]

German Stem Cell Network Conference Poster Prize, Heidelberg, Germany [2014]

Nucleic Acids Research Poster Prize, Heidelberg, Germany [2014

]Dresden International Graduate School Travel Award, Dresden, Germany [2012]

Future of Science Foundation Scholarship, Keystone Symposia, Utah, USA (2012)



Debojyoti Chakraborty
Jun 28
Hello everyone,

we have successfully used NgAgo in HeLa cells following a protocol where we used 700ng NgAgo plasmid and 500 ng gDNA (non purified, heat inactivated PNK). We confirmed knockout of GFP from a plasmid and could see the effect even after 96h. About 40% reduction in Western blot could be seen although there is scope for more optimization.

cheers
debo


Debojyoti Chakraborty
Jun 29
Other recipients: o.ku...@gmail.com, crisp...@gmail.com
Hi Ilasadar,

We did not use the Addgene plasmid but a plasmid sold by miaolingbio (miaolingbio.com). It's called NLS-NgAgo-pCDNA3.1. It has a CMV promoter which can be shutdown in certain types of cells (like ES cells) so putting in a CAGGS might help in expressing the protein more.
Our protocol used lipofectamine 3000, 500ng gDNA and 700 ng plasmid. The gDNA was phosphorylated with PNK but was not purified. We have done it once to check for GFP knockdown (co-transfected GFP plasmid) so we need to verify with more controls/sequencing which we are currently doing.

hope that helps
debo


Debojyoti Chakraborty
Jun 28
Hi guys,

We have used this system and it works. We have followed the same protocol as mentioned in the paper and were able to confirm knockout both on FACS as well as western. We purified the oligos on gel after T4 PNK and saw the phenotype after 48 and 72 h.

cheers
debo


Debojyoti Chakraborty
Jun 30
Other recipients: zhouya...@163.com
Here's the protocol we followed:


We first phosphorylated the gDNA using the PNK kit from Ambion for 1 hr followed by heat inactivation at 95 degrees for 5 min. We then went on to transfect 100,000 cells in 12 well plates as follows:

Mix 1:
Lipo reagent- 3ul
Optimem- 43ul
EGFP N1- 1 ug
NgAgo- 900 ng
gDNA- 500 ng

Mix 2:
Lipo 3000- 2ul
Optimem- 48ul

Mix 3:
Media- 250ul
Optimem- 150ul


Mix1 and Mix2 was incubated for 10min each and then both were mixed together and incubated at RT for 30min

This was now added to cells. To each well, 50 ul of Mix 3 was added on top. No further media was added to the cells.
After 12h, the mix was removed and fresh media was added.


Jan Winter
Jun 28
Hi guys,
Just had a couple of tests with ngago from addgene and custom Oligos,  so I did not try to reproduce the paper.
And in my hands I see an effect that is slightly less than using the same target sequence region using the px459, but it works.
I phosphorylated  the oligos  using T4PNK and transferred 300 ngago per 24 well to hek293t cells.

Cheers
Jan

Haiyang Yu
Jun 28
Hi, Jan,
It is exciting that you got it work! Could you post a detailed protocol? Please include how you did T4PNK, after that did you purify your oligos? How did you transfect and how long did you wait?
Thank you!
Jan Winter
Jun 30
Hi guys,
okay some more stuff from my side, a detailed protocol will come once I did some more tests, so fare I have NO INDEL DATA.
What I did:
- 24 bp oligos
- phosphorylated them using T4 PNK (standard reaction, 300 pM of oligo) for 1 hr at 37 with T4DNA Ligase buffer
- cleaned up oligos using Qiagens Nulceotide removal kit for 4x300 pM of oligo per column
- measured with Qubit ssDNA kit (but I have no clue about the phosphorylation)
- transfection of  50000HEK293T cells (24 well plate, 0.5 mL per well, 50000 cells per well, 2 uL TransIT LT1 reagent) with Mirrus TransIT LT1-> 500 ng of ngAGO from Addgene and 300 ng of oligo
- I do not change any medium, but I add another mL of complete grwoth medium 24 hr after transfection
- FACS assay after 72 to 96 hr
My HEK cells contain a custom reporter as well as Cas9 expression, which is linked with eGFP by a P2A sequence.
My observations were the following:
- in presence of ngAGO transfection I see a slight reduction of eGFP (even without any oligo) in the range of 3-5 %
- I see no effect if I transfect 24 bp oligos alone without ngAgo
- I made custom oligos against specific regions of my reporter and target genes of my reporter systems -> I see an effect in FACS wich is only present if I transfect ngAgo + Oligos together
- The effect is slightly worse than transfection my sgRNA plasmid (cells are Cas9 positive)
- If I compare the phenotypic effect of sgRNA vs ngAGO oligos (that target the same region), ngAgo is less effective
I will do some tests next week and will also have a look at indel formation in the next month.




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