I am about to start some new experiments that rely on TAT-Cre (from Millipore) to delete a gene of interest in cell culture in order to study the impact on cell differentiation and gene expression. I will also perform ChIP. Initially, I will be optimising the concentration of TAT-Cre needed to limit toxicity.
Does anyone have any recommendations or things to be aware of when working with TAT-Cre? Thanks.
Hello Jie
I did not order the one from Millipore, but used a TAT-Cre produced by our Biochemistry Department. It worked quite well! You will get a lot of cell death, which is also indicative of successful entry of TAT-Cre into the cells. I found that serum in the medium limits the toxicity, but this is consistent with the peptide being less efficient in the presence of serum. I decided to stick to serum to promote cell survival, whilst increasing the concentration of TAT-Cre. You can also play with the time of exposure. I worked with primary mouse vascular smooth muscle cells and treated them with 4 uM TAT-Cre for 16h, which resulted in ≅ 80% gene editing efficiency.
Hope this helps.
Thank you Andrea. Any thoughts on toxicity and applied concentration?
Hello Jie
I did not order the one from Millipore, but used a TAT-Cre produced by our Biochemistry Department. It worked quite well! You will get a lot of cell death, which is also indicative of successful entry of TAT-Cre into the cells. I found that serum in the medium limits the toxicity, but this is consistent with the peptide being less efficient in the presence of serum. I decided to stick to serum to promote cell survival, whilst increasing the concentration of TAT-Cre. You can also play with the time of exposure. I worked with primary mouse vascular smooth muscle cells and treated them with 4 uM TAT-Cre for 16h, which resulted in ≅ 80% gene editing efficiency.
Hope this helps.
Glad it was helpful!
When I cultured the cells in CDM, the viability was pretty low regardless of the concentration (
Hello Ralitsa!
I'm working with primary mouse vascular smooth muscle cells too. Have you tried to treat TAT-Cre followed by chloroquine like these guys http://jcb.rupress.org/content/183/3/409.full#sec-7 ? How do you analyse the efficiency? Do you use qRT-PCR analysis of TAT-CRE treated clones? How many days after treatment individual colonies need to grow enough size?
Thanks!
Hello guys!!
I am using TAT Cre on mosue lymphocytes. I am really struggling with the viability issues. Is someone having any experience with T cells ?
Also, after the TAT Cre treatment which condition do you put the cells under
anti-CD3? IL-7? or what ?
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