A note on our IRE1 knockout mammalian cells

A note on our IRE1 knockout mammalian cells:

Mouse embryos lacking IRE1α (in the strain background we had explored) die between embryonic day 9.5 and 10.5, likely due to a defect in placental development (1, 2). Though we were able to culture adherent cells from dissociated IRE1α-/- embryos and eventually to immortalize these cells with SV40 T antigen (3, 4), 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 meaningful way to create a comparison group to the knockout cells is to rescue them with an IRE1 transgene. We had done something similar, rescuing the mutant cells with IRE1β, but the restoration of IRE1 function was rather partial (4). More recently, Romain Volmer from our lab has rescued these cells with IRE1α and substantially restored XBP1 splicing (5).

David Ron

Cambridge, 12 September 2013

REF:

1. Ghosh R, Lipson KL, Sargent KE, Mercurio AM, Hunt JS, Ron D, et al. Transcriptional regulation of VEGF-A by the unfolded protein response pathway. PLoS ONE. 2010;5(3):e9575. 

2. Iwawaki T, Akai R, Yamanaka S, Kohno K. Function of IRE1 alpha in the placenta is essential for placental development and embryonic viability. Proc. Natl. Acad. Sci. U.S.A. 2009 Sep 29;106(39):16657–62. 

3. Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, Harding HP, et al. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science. 2000 Jan 28;287(5453):664–6. 

4. Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP, et al. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature. 2002 Jan 3;415(6867):92–6.

5. Volmer R, van der Ploeg K, Ron D. Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains. Proc. Natl. Acad. Sci. U.S.A. 2013 Mar 19;110(12):4628–33.