21 August 2017 

A note on the use of cell lines lacking UPR components:

Over the years our lab has produced a number of cell lines lacking genes involved in the UPR and ISR. We are happy to see them used in research and place no restrictions in that regard. To the extent possible, we have deposited some of the most popular lines with the ATCC and these are available for use – though the associated cost is staggering, in my opinion. Unfortunately, depositing lines with the ATCC is enormously labour intensive and some of our lines (the IRE1a_KO, GADD34_KO, ATF4_KO immortalized MEFS) are therefore not available for distribution via the ATCC. We are happy to make these available to all academic researchers (no strings attached), but given the work and cost associated with sending cells, we need to first be convinced that the cells we send are going to be of use to the lab requesting them and that this is an efficient use of everyone’s time.

In that vein, some considerations follow:

We are unable to meet the demand for I˚ MEFs. Therefore the cells we send out and those deposited at ATCC are SV40 large T-antigen transformed.

Please note that immortalized cells have undergone lengthy adaptation to tissue culture and some phenotypic features, such as the hypersensitivity to ER stress may have been attenuated. As a consequence wildtype transformed MEFS are a poor control for the analysis of complex phenotypes in the mutant cells. This is true even if the parental stock of wildtype and mutant MEFS were highly comparable (i.e. even if the two cell populations were isogenic at their origin). The only thing that can be stated with confidence in regards to these knock-out cells is that they lack the gene in question. Thus they may be useful loss-of-function model for proximal signalling events that cannot be easily compensated for by adaptation in function and activity of other genes (trans-suppression).

This problem is compounded further in case of the IRE1a_KO cells as described in the brief document available at this link.

Over time potent inhibitors of PERK and IRE1 have been discovered and are commercially available. 4µ8C, the IRE1 RNase inhibitor, discovered in our lab, is commercially available from Tocris bioscience and the PERK inhibitor we helped characterize, GSK2606414, is now available commercially from Millipore Calbiochem (Cat. 516535). These reagents have off target effects, thus a phenotype elicited by them in cells cannot, with any certainty, be attributed to PERK or IRE1 inhibition. However, when applied at a high enough concentration these compounds are able to completely block the RNase activity of IRE1 and the kinase activity of PERK. Thus, if application of 4µ8C or GSK2606414 does not block the phenomena attributed to IRE1 or PERK, the hypothesis can be rejected. 

Because inhibitors are portable and relatively inexpensive, this kind of experiment is very powerful: often it can be conducted in a cellular system that has already been developed to study the process in question (something that might not be possible in the knockout MEFs).
Furthermore, it is worth pointing that CRISPR-Cas9 technology has developed to the point where many labs can now inactivate genes in their cell of choice. For example, we have inactivated IRE1a, GADD34, GCN2 & FICD in CHO cells, for reasons of convenience. It is therefore worth pausing to consider the relative effort and cost associated with inactivation of the gene of interest in a reliable and well studied cellular system and compare that with the potential benefits of the KO MEFs.

David Ron Cambridge, UK