Hi,
I generated a crispr ko cell line. The protein was more than 90% knocked out. But, now after several months od thawing and repassaging,the protein seems to comeback.
I plan to do a grloth rate study with wild type and ko cells in mice but do not know what will happen now. The old ko cells are growing slowly and would take time
Regards,
Pratik
Hi,
Can you write more details which protein for example and so on.
Hi Pratik,
I guess some WT cell remained as a "contamination" in your KO stock. If these WT cells grow more efficiently that the KO (and even if they are not more efficient) they will be adding more WT protein, including your problem protein, to the total cell mass. That's why you may be starting to see the this protein coming back.
How did you isolate the KO strain in the first place?
Cheers,
The knock out cells do not express the protein. The protein that it appears to come back is from the contaminating cell types that were not gene deleted. Since your knockout cells do not proliferate very well, with time your contaminating cells could increase in population due to proliferation and ultimately dominate the knockout cells. Go for a method to purify the knockout cells or can discard these ones and always use the old/early passage ones that are dominated by the knockout population.
If you knock out the gene - gene for protein has been deleted-then the protein expression has gone, but if it has been only inactivated say some part of gene deleted ot mutated , truncted protein will be express but may be not functional any more.
Hi,
My answers:
1. Protein is GLUT1 (basal glucose transporter present in entire human body which is responsible for glucose uptake in tissues like brain and heart).
2. I generated KO clones via routine CRISPR methology. I used gRNA plasmid to transfect cells and then I did seed cells in 96 well plate, then 24 and 6 well plate. After that I performed western blot see which clones have lowest expression of GLUT1, Out of my 12 clones from 96 well plate (10 had nearly 90% inhibition or more and 2 had about 75-80%).
3. I can now see changes in morphology of my KO cells. They have much larger protruding podias (in comparison to smaller podias of A549 cells). If someone sees those cells, they can easily tell the difference. I would actually put a picture of those cells with WT cells soon.
4. I have not done any growth curve with these cells but they appear to grow at almost the same rate as WT cells. Like started with 1 million cells vial of each (WT ad KO) on the same day.
I thawed a fresh vial of KO and WT cells.
a. WT - day 1- 1 million cells; Day 3 - 5 million ; day 6 - 3 3 million cells
b. KO - Day 1 - 1 million cells; day 4 - 4 million cells; day 7 - 29 million cells
If you compare the two, you can say that after 4 days there were more WT cells but if you look at growth rate after 4 days, KO cells are outpacing the WT cells. I am worried about this phenomenon.
Any suggestions on isolating KO population?
Regards,
Pratik
Dear Pratik,
From your explanation it may be the case Nico Merola
mentioned above when your 10 isolated clones are KO for GLUT1 only in one allele. Could you Check GLUT1 mRNA levels by RT-PCR to help you select clones?You could also transfect these 10 clones again with your Crispr approach (not the most recommended since it may increase off-targeting) in an attempt to target the intact allele in these clones and re-do the selection.
Best,
I may generate Glut3 and Glut4 ko clones in future. What startegy do you recommend for knocking out both alleles.
One question, if one allele is knocked off and other is still active, how come I have only 10% protein expression. It should 50%.
Yes. If it is one allele that is deleted, the other allele should result in the expression of 50% of the total copy numbers. However, we do not know how many cells in your culture is completely gene deleted and how many of them were 50%. It is possible that your cells are a heterogeneous population some of them do not express while the others may have 50% expression and the total extraction and quantification may have the representation of 10% when combining all the cells together. Try to make some single cell clones and examine individual clones for the actual % loss in each of the sub-clones.
In this way you can track what is going on.
I have other clones that also have 10% expressions. I would grow these for my further studies. Btw, I have injected mice with Ko and wt cells. Lets see.
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