Please send me information on dCas9-KRAB or AICS-90 cells as well as on vectors expressing dCas.
We seek viral vectors and/or expressing iPSC.
Thank you in advance, Ruth
Hi Ruth. You can find the QC data and other information about our dCas9-KRAB line at https://www.allencell.org/cell-catalog.html--simply scroll through the list to AICS-0090, and click on it. There is also a link at the top of the AICS-0090 page to order the hiPSC line from Coriell, or the plasmids from Addgene.
Hi,
I have a further question related to the AICS-0089 cell line which similarly expresses the dCas9-KRAB construct. We have previously obtained the AICS-0089 cells, and we would like to utilise the neomycin resistance (NeoR) cassette within the genomic locus with the dCas9-KRAB construct to enable selection for cells with higher dCas9-KRAB expression. I am wondering if you can advise whether neomycin, or perhaps an analog such as Geneticin, has been used in the generation of this line, and if so whether you are able to advise on a concentration of neomycin that was used during this?
Many thanks,
Sarah
Hi Sarah,
Thank you for asking. G418 was not used in generating that line as we typically avoid the use of chemical selection agents during development of our cell lines. However, G418 selection is effective in general with WTC hiPSCs, and we recommend this protocol (from CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs - PMC) as a starting point. This cell line’s genetic elements were conserved for these purposes, although we do not have experience with the precise experiment proposed. All of our G418 selection experiments have been done with transgenes introduced to AAVS1, instead of CLYBL.
As an aside, we were wondering about your interest in increasing expression. Is knockdown of some genes insufficient?
Best,
Janani