massive cell death after transfection
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Phil Abbosh
May 1, 2015, 6:02:20 PM5/1/15
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Guys
Quick question. i designed guides to model a translocation. i transfected the guides cloned into pX330 into 3T3 cells, then unfortunately was traveling for 3 days. when i harvested cells for gDNA, all of the guides i designed against either of two introns for gene A (two guides per intron) caused ~100% cell death. I also transfected 5 other guides the same day and had >90% cell survival after transfection (which is pretty normal for my protocol). is there any other conclusion other than "the guides are super-efficient and loss of the last couple exons of gene A (a growth factor receptor) is lethal"? super-high efficiency in all 4 spacers just seems too good to be true but it seems more than coincidental that those 4 all cause the same result.
i am going to repeat next week and harvest a day or two earlier.
phil
Quick question. i designed guides to model a translocation. i transfected the guides cloned into pX330 into 3T3 cells, then unfortunately was traveling for 3 days. when i harvested cells for gDNA, all of the guides i designed against either of two introns for gene A (two guides per intron) caused ~100% cell death. I also transfected 5 other guides the same day and had >90% cell survival after transfection (which is pretty normal for my protocol). is there any other conclusion other than "the guides are super-efficient and loss of the last couple exons of gene A (a growth factor receptor) is lethal"? super-high efficiency in all 4 spacers just seems too good to be true but it seems more than coincidental that those 4 all cause the same result.
i am going to repeat next week and harvest a day or two earlier.
phil
YIng Dang
May 1, 2015, 7:20:28 PM5/1/15
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Super interesting! Another possibility is endotoxin contamination in those constructions. make sure you update here with your repeat result;)
Ying
Guillaume REQUIER
May 1, 2015, 7:41:36 PM5/1/15
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Hi, what is the receptor ?
Anoeska
May 4, 2015, 7:34:30 AM5/4/15
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Did you do control transfections like pX330 without any inserted gRNAs or 'no DNA'? It can also be something of the transfection itself that is not tolerated by the cells.
Anoeska
Op zaterdag 2 mei 2015 00:02:20 UTC+2 schreef Phil Abbosh:
Op zaterdag 2 mei 2015 00:02:20 UTC+2 schreef Phil Abbosh:
Phil Abbosh
May 4, 2015, 1:54:47 PM5/4/15
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Thanks for the responses everyone.
Anoeska. i do GFP alongside the guide constructs and that was fine. i use that as the negative control template for detection of indels. Also, the other 4 guides i tested that same day did not show toxicity, nor any of the 15-20 i have tested before that.
similarly Ying, I didnt get toxicity with other plasmids prepped in the same batch and transfected on the same day, so i think endotoxin is less likely, although a good thought.
phil
Anoeska. i do GFP alongside the guide constructs and that was fine. i use that as the negative control template for detection of indels. Also, the other 4 guides i tested that same day did not show toxicity, nor any of the 15-20 i have tested before that.
similarly Ying, I didnt get toxicity with other plasmids prepped in the same batch and transfected on the same day, so i think endotoxin is less likely, although a good thought.
phil
JP
May 4, 2015, 9:08:05 PM5/4/15
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Even if all the cells die, you can still harvest them to look for cuts.
Try transfecting your constructs alongside a cDNA expression vector (with silent mutations to avoid being cut by your gRNA) to rescue the cells.
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