CBh promoter in mESC

[grzegorz...@gmail.com]

Hello All,

Has anyone tried the Zhang's lab plasmids in mESC? is CBh promoter working there nicely?

thank you in advance

~greg

[Marc Morgan]

I am also curious about this. One publication shows an EF-1a promoter plasmid which should be active in mESCs. CBh looks similar to the chicken actin-CMV hybrid (pCAG) which is highly active in ESCs, but I would certainly be curious to know if anyone has used the CBh-Cas9 plasmid in mouse ESCs. 

[Feng Zhang]

Hi Marc, CBh works well in mESCs. Good luck!

Feng

--
Feng Zhang
Core Member, Broad Institute of MIT and Harvard
Investigator, McGovern Institute for Brain Research
Keck Career Development Assistant Professor of Neuroscience and Biological Engineering, MIT
zha...@mit.edu

On Fri, Jun 14, 2013 at 11:18 AM, Marc Morgan <marcmo...@googlemail.com> wrote:

I am also curious about this. One publication shows an EF-1a promoter plasmid which should be active in mESCs. CBh looks similar to the chicken actin-CMV hybrid (pCAG) which is highly active in ESCs, but I would certainly be curious to know if anyone has used the CBh-Cas9 plasmid in mouse ESCs. 

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[Zhi ZHOU]

Hi Feng, 

Im also interested in the expression kinetics of CBh promoter when compared to CAG, CMV, EF1a. (in mESC, hES/iPS)

Im looking forward to getting your post!

Thank you.

Zhou

2013年6月15日土曜日 4時44分45秒 UTC+9 Feng Zhang:

[Feng Zhang]

Hi Zhou,

We have not yet done a systematic comparison of CBh, CAG, CMV and EF1a. Nevertheless CBh works well for us in ES cells. It is about the same strength or slightly weaker than EF1a. Below is the paper describing it:

http://www.ncbi.nlm.nih.gov/pubmed/21476867

Hope this helps!

Feng

[Zhi ZHOU]

Hi Feng, 

Thank you for your quick response, and suggestion.

>the same strength or slightly weaker than EF1a

Then, the benefit using CBh promoter seems to be only its small size........

Anyway, thank you so much again!!

Zhou

2013年8月30日金曜日 12時38分33秒 UTC+9 Feng Zhang:

[lasky...@gmail.com]

Hi  Feng, does CBh works well in hESC? Thank you!

       

在 2013年6月15日星期六UTC+8上午3时44分45秒，Feng Zhang写道：

[jing lin]

Hi, Feng,

     Do you know that CBh promoter works well in bone marrow cells, especially hematopoietic stem cells?  Thanks!

Jing

[Feng Zhang]

Dear Jing,

We have not tried CBh in HSCs... Perhaps someone else on the forum will be able to answer?

Best,

Feng

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[Ayal Hendel]

Did anyone get the PX330 (CBh) or any other vector to work in Human hematopoietic stem cells?

Thanks,

Ayal

[a.maiques]

Hi all, 

I've tried to nucleoporate the PX461 vector, also driven by CBh, into THP1 cells (human monocitic leukemic cell line) without any GFP positive cells. 

I've used Amaxa 4D-system and had very nice GFP+ cells in the positive control.

It may well be that this promoter is not functioning in the hematopoietic cells (or that nucleoporation didn't work at all with the plasmid either)

Has anybody has other results about this?

thanks, 

Alba 

[crispr.cas...@gmail.com]

Dear Feng,

Can Cbh work in human ESCs or hiPSs? 

Best,

Ming

在 2013年6月14日星期五 UTC+2下午9:44:45，Feng Zhang写道：

[crispr.cas...@gmail.com]

Dear Feng,

Cbh promoter is not suitable for the over-expression of Cas9 in human Embryonic Stem cells or human iPS cells.  But in the Nature Protocol, what you wrote is:

hesc (Hues 9) culture and transfection ● tIMInG 3–4 d
 crItIcal hESCs and human induced pluripotent stem cells can vary widely in their transfection efficiency, tolerance of single-cell dissociation and maintenance conditions. For a given cell line of interest, relevant literature or the distributor should be consulted.

30| Maintaining HUES9 cells. We routinely maintain HUES9 cells (a hESC cell line) in feeder-free conditions with mTesR1 medium. Prepare mTeSR1 medium by adding the 5× supplement included with the basal medium and 100 μg ml−1 Normocin.

31| Prepare a 10-ml aliquot of mTeSR1 medium supplemented further with 10 μM ROCK inhibitor.
32| Coating a tissue culture plate. Dilute cold GelTrex 1:100 in cold DMEM and coat the entire surface of a 100-mm tissue

culture plate.
33| Place the plate in an incubator for at least 30 min at 37 °C.

34| Thaw a vial of cells at 37 °C, transfer the cells to a 15-ml Falcon tube, add 5 ml of mTeSR1 medium and pellet at 200g for 5 min at room temperature.

protocol

35| Aspirate the GelTrex coating (Step 32) and seed ~1 × 106 cells with 10 ml of mTeSR1 medium containing ROCK inhibitor from Step 31.

36| Replace with mTeSR1 medium without ROCK inhibitor after 24 h and refeed daily.
37| Passaging cells. Passage the cells before they reach 70% confluency.
38| Aspirate the mTeSR1 medium and wash the cells once with DPBS.
39| Dissociate the cells by adding 2 ml of Accutase and incubating them at 37 °C for 3–5 min.
40| Add 10 ml of mTeSR1 medium to the detached cells, transfer the mixture to a 15-ml Falcon tube and resuspend gently. 41| Replate the cells onto GelTrex-coated plates in mTeSR1 medium with 10 μM ROCK inhibitor.

42| Replace with normal mTeSR1 medium 24 h after plating.
43| Transfection. We recommend culturing cells for at least 1 week after thawing and before transfecting by using the Amaxa

P3 primary cell 4D Nucleofector kit.
44| Refeed log-phase growing cells (50–70% confluency) with fresh medium 2 h before transfection.

45| Dissociate to single cells or small clusters of no more than ten cells (as viewed under the microscope) with Accutase and gentle resuspension.

46| Count the number of cells needed for nucleofection (200,000 cells per transfection) and spin down at 200g for 5 min at room temperature.

47| Remove the medium completely and resuspend it in 20 μl of S1-supplemented P3 nucleofection solution, per 2 × 105 cells. 48| Pipette the resuspended cells with added DNA (Steps 9 and 19) into electroporation cuvettes and electroporate

according to the suggested program. For 2 × 105 cells, we typically use 1 μg of total DNA.
49| Gently plate the electroporated cells onto coated 100-mm plates supplemented with 10 μM ROCK inhibitor.

50| Check transfection success (Steps 11 and 28) and refeed the cells daily with regular mTeSR1 medium beginning 24 h after nucleofection. Puromycin selection can be applied at a concentration of 0.5 μg ml−1 (may vary depending on the cell line). Typically, we observe >70% transfection efficiency with Amaxa nucleofection. 

It seems that you use a plasmid pSpCas9(BB)-2A-Puro(Px459-Cbh promoter) to nucleofect the human ES cells, AND then you add 0.5ug/ml Puromycin to select the cells resistant to puromycin. It would be great if you can explain how did you guys make the Cbh promoter work in human ES cells?

Best,

Ming

在 2014年8月31日星期日 UTC+2下午5:08:43，Feng Zhang写道：

[Fei Ann Ran]

Hello Ming,

If your question is whether the Cbh promoter works in human ES cells, then we have indeed had success using it in several HUES cell lines following standard transfection protocols (either Fugene lipofection or nucleofection). However, as Feng mentioned earlier we've not done a systematic comparison with other promoters in hESC or HSCs so it's possible that other promoters may work as well or better.

Best,

Ann

[crispr.cas...@gmail.com]

Thanks a lot, Ann. 

Following are some words from a student's thesis, I'm confused who is right. 

iPSCs are a very difficult cell type to experiment with and choosing the optimal iPSC promoter is no exception. The original Cas9 plasmid contains only a Chicken β-Actin Promoter (pCBh). This promoter has been shown to express proteins only in normal cell types (i.e. HEK293t cells) but will not drive expression of proteins within hESCs or iPSCs. A suitable replacement for the pCBh promoter is a CAG promoter. The CAG promoter consists of three genetic components that will help drive expression of proteins within a hESC. Its components include a Cytomegalovirus (CMV) enhancer, a Chicken-β-actin promoter, and a Rabbit β-Globulin Splice acceptor gene. Previous studies have shown that the stronger CAG promoter will help promote expression of transgenic genes within hESCs (Chen et. al., 2011). 

There are many labs using the pSpCas9-(BB)-2A-Puro(Px459) plasmid. They want to select the cells which can be resistant to puromycin. But after adding puromycin, even at 0.3ug/ml(you use 0.5ug/ml), 24hours later, all of the cells will die. It seems that the Cas9-Puro cannot be expressed at all. Yeah, maybe you will say that they are not careful enough, but I don't think so. 

Best,

Ming

在 2015年2月6日星期五 UTC+1下午6:31:46，Fei Ann Ran写道：

[Fei Ann Ran]

Hi Ming,

Have you tried PX330 or PX458 (Cbh::SpCas9-2A-GFP) in your cells to check for expression? It's possible that the issue you have is related to the Puro cassette in PX459 (rather than Cbh), which some people are having issues with. If that's the case I'd be happy to send you a new PX459.

Ann

...

[crispr.cas...@gmail.com]

Hi Ann,

I also tried PX458 in hiPS, less than 1/10000 cells were GFP positive. I used pmax-eGFP to make sure that our nucleofection could work properly, here 80-90% cells were GFP positive. 

One of my friends sequenced the PX330, they found some mutations. Finally they used another Cas9 plasmid from another lab.

Best,

Ming

在 2015年2月6日星期五 UTC+1下午9:20:07，Fei Ann Ran写道：

[alba maiques]

Hi again, 

just to update my previous email, 

i've been able to sort THP1 cells transfected with the PX461 vector, however the promoter seems to have very weak activity, less than 0.5% of the cells were GFP+.

probably using other promoters to drive the expression will work better for haematopoietic cells..

alba. 

--

[christop...@univ-paris-diderot.fr]

Hi everybody,

we tried to transfect, by amaxa 4D,  the PX458 plasmid into human ESC and we have only 15-20% of GFP expression 24h post transfection.
I think Cbh promoter is not the best choice for this cell type.
Feng, have you planned to do a new version of PX458 and PX459 with "hESC promoter" like PGK or EF1 alpha ?

Thanks

Christophe

[Albert Ruzo]

In my opinion, if you have some cells expressing GFP, then the promoter is working. The problem may be the transfection efficiency, and yes, for such a big plasmid as pX458/9 the transfection efficiency is pretty low in hESC. 

On Feb 11, 2015, at 3:48 AM, christop...@univ-paris-diderot.fr wrote:

Hi everybody,

we tried to transfect, by amaxa 4D,  the PX458 plasmid into human ESC and we have only 15-20% of GFP expression 24h post transfection.
I think Cbh promoter is not the best choice for this cell type.

Feng, have you test differents promoters in the same backbone to compare GFP expression in hESC ?
Have you planned to do a new version of PX458 and PX459 with "hESC promoter" ?

Thanks

Christophe

Le mardi 14 mai 2013 12:32:00 UTC+2, grzegorz...@gmail.com a écrit :

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[guoji...@gmail.com]

Hi Feng,

I am wondering if pX330 plasmid can be used in mosquito cell lines (Aede. albopictus). I am planning to generate some mutants from these cell lines. I know pX330 is developed for mammalian cells, so I am not sure whether the promoter CBh for Cas9 and U6 for sgRNA can be efficiently expressed in mosquito cells, although some papers have done some experiments, but not related to pX330. 

Thanks.

Jinchao Guo

PhD student, 

Faculty of Biological Sciences,

University of Leeds

[Feng Zhang]

Hi Jinchao,

Thank you for your email. Unfortunately I'm not aware of any experiment that have tested the CBh promoter in mosquito. I would suggest using a more traditional insect promoter for your experiment in mosquitos.

Good luck with your experiment!

Feng

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