A note on our IRE1 knockout mammalian cells:

Mouse embryos lacking IRE1a (and those lacking IRE1a & IRE1b) die at about ED10. Though we were able to culture adherent cells from dissociated embryos of the IRE1a-/- (and compound embryos lacking both IRE1a & Ire1b) and eventually to immortalize these cells with SV40 T antigen, the efficiency of the immortalization procedure was very low and the population of cells went through a bottle neck at that point. Therefore, we know next to nothing about the origin of the cells that were immortalized and they cannot safely be referred to as MEFS. By the same token, we have no wildtype comparison cell line to offer that can be used for physiological studies.

In our opinion, the safest way to create a comparison group is to rescue the mutant cells with an Ire1a transgene. We had done something similar, rescuing the mutant cells with Ire1b, but the restoration of IRE1 function was rather partial (Calfon, et al., 2002, Nature; 415:92) and I suspect that the rescue procedure may not be that easy with the more potent IRE1a.

David Ron

NYU, April 8, 2008