The small co-chaperone p23 overexpressing transgenic mouse

Abstract

Studies from multiple laboratories have identified the roles of several ER stress-induced cell death modulators and effectors. Earlier, we described the role of p23 a small co-chaperone protein in preventing ER stress-induced cell death. p23 is cleaved by caspases at D142 to yield p19 (a 19 kDa product) during ER stress-induced cell death. Mutation of the caspase cleavage site not only blocks formation of the 19 kDa product but also attenuates the cell death process triggered by various ER stressors. Thus, uncleavable p23 (p23D142N) emerges as a reasonable candidate to test for potential inhibition of neurodegenerative disease phenotype that features misfolded proteins and ER stress. In the present work we report the generation of transgenic mouse lines that overexpress wild-type p23 or uncleavable p23 under the control of a ROSA promoter. These mice should prove useful for studying the role of p23 and/or uncleavable p23 in cellular stress-induced cell death.

Fig. 1

Schematic diagram of the constructs used for generating the transgenic mice. N-terminal Flag-p23 (human) and Flag-p23D142N were each subcloned into a pcDNA3 expression vector (driven by the ROSA promoter) by PCR-amplification of each construct. Purified linearized DNA (1–2ng/ul) was injected into the pronuclei of fertilized oocytes derived from C57BL/6 X C57BL/6 mice. WTp23 (p23wt) and p23D142N (p23unc) transgenic mice were produced by standard techniques as mentioned in METHODS

Fig. 2

p23 transgene from tail DNA of mice. Tail DNA from mice was isolated and PCR was performed using the primers as mentioned in Methods. (A) Shown is a representative gel scan demonstrating the p23 transgene from tail DNA of mice. Five p23wt and three p23unc transgenic founder mice were chosen for establishing the transgenic lines. (B & C) Western blots showing the p23 expression in p23wt and p23unc mice. p23 protein was detected using anti-FLAG antibody. GAPDH served as a loading control.

Fig. 3

p23 staining by immunohistochemistry. Fluorescence immunohistochemistry on brain sections from p23wt or p23unc mice shows widespread p23 expression (green) in cortex and thalamus but not in the striatum. Staining is seen perinuclearly in most cells and in some cells bodies in all areas of the brain. Non transgenic (Ntg) mice showed low or no background staining. DAPI (blue) was used to stain nuclei.

Fig. 4

Expression of p23 in different tissues: Western blots showing expression of p23wt and p23unc protein in brain (B), liver (L) and heart (H) from transgenic (Tg) mice as compared to Non transgenic (Ntg) controls. p23 protein was detected using anti-FLAG antibody. GAPDH served as loading controls.