Dear all,
I constructed a mutant 4T1 cell line using CRISPR and implanted it into the 4th mammary gland of BALB/c recently. The tumor grows well at the same rate as WT in the first 12 days but 2 weeks later some of the CRISPR-modified tumor regressed while some remains small about 4~5mm long. I have some guesses but I really need you guy's assitance to help me out.
1. Maybe it's just a good thing, a phenotype. The presence of my gene ruins the immunosurvilence so the mutant cells are attacked and ruled out by the immune cells.
2. The contamination of Mycoplasm. But I think the contaminated cells cannot grow into a tumor at all. I don't have so much experience, so any information about this is appreciated.
3. The loss of cell viability. You know, it takes almost 2 months to finish the entire work of CRISPR. So I am afraid the 4T1 invasiveness will decrease. But for the same reason mentioned in 2, I think if viability is an issue the tumor will not appear at all. I need you guy's help to give me any experiences about this.
Thanks in advance for all your time and kind help, guys!
Hi Lai,
I think it's important to know what kind of mutant you generated using CRISPR and also how you did it: Did you do a knockout/interference experiment or did you induce a certein mutation? Did you then use single cells or the bulk that survived (possible) selection. What did you expect to happen from interfering with this gene? How good are your guide - are you observing off-target effects (i.e. the knockout of vital genes)?
point 2. and 3. could be tested by running controls, i.e. going through the whole procedure of your CRISPR experiment without guides/control guides and then also inject them into your mice.
Hope that helps
Hi Thomas,
Thanks for your response! It's really helpful. It's a knockout single cell line where CRISPR introduces a one dp deletion at the DSB. I confirmed it by cloning the PCR pruducts into construct and by sequencing 4 colonies. Yes, tumor regression is one of our hypothesis of mutating this gene. Right now, I have one negative single colony from the CRISPR screening, so I plan to inject that one as a control. However, we haven't tested the potential offtargets but I have other two colonies with different mutation so I will inject them to confirm our phenotype. With these at hand, I still want to know what will happen if we use an aged cell line for xenograft. Do they not grow at all? Because only some of our KO cell line xenografts regressed but some still remain just smaller. I want to know can I rule out the possibility of passaging too many times by observing this phenomenon.
Thanks again and sincerely for your kind help!
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