- Meeting abstract
- Open Access
PW03-024 - A transgenic mouse model for variant procaspase-1
© Hermsdorf et al; licensee BioMed Central Ltd. 2013
- Published: 8 November 2013
- Bacterial Artificial Chromosome
- Mouse Oocyte
- Transgenic Mouse Line
- Cytometric Bead Array
- Foster Mother
We have detected several genetic variants of CASP1 in patients suffering from unexplained recurrent febrile episodes. Paradoxically, in vitro and in vivo analyses of patients’ cells revealed decreased enzymatic activity of these caspase-1 variants leading to impaired cytokine production despite the proinflammatory phenotype of the patients. The pathophysiological processes associated with CASP1 variants are still under investigation.
In order to recapitulate the effects of the CASP1 mutations found in the patients we tried to establish a bacterial artificial chromosome (BAC) transgenic mouse line expressing enzymatically inactive Casp1 C284A under the control of the own promoter.
The purified BAC fragment containing Flag-tagged Casp1C284A (Casp1C284AFlag) was injected into the pronuclei of fertilized C57Bl6 mouse oocytes, followed by transfer of these oocytes to pseudopregnant foster mothers. Pups born from these mothers were analyzed for the presence of full-length Casp1C284AFlag by screening with sequence specific PCR, Southern blot, and sequencing of the transgene. Casp1C284AFlag transgenic mice were crossed to conventional Casp1 knock-out (KO) mice and the immunological phenotype of the progeny was analyzed by in vitro stimulation of BMDCs. Expression levels of the Casp1C284AFlag transgene were quantified by qRT-PCR and Western blots. Released cytokine levels were determined by cytometric bead arrays.
From two independent pronucleus injections we received 180 pups. Only three of them harbored transgene sequences and only one female animal proved to harbor the complete Casp1 C284AFlag transgene (TG). Crossing to Casp1 KO mice yielded the following genotypes: Casp1 WT/WT/TG, Casp1 WT/KO/TG, and Casp1 KO/KO/TG. qRT-PCR analyses revealed that unstimulated Casp1 C284AFlag transcription was reduced to 0.1% of wild-type Casp1. Hence, protein expression could not be detected in unstimulated cells. However, stimulation with LPS upregulated transcription and low-level translation of Casp1C284AFlag in BMDCs. Determination of released cytokines after LPS/ATP stimulation revealed increased release of IL-6 and TNF-a from Casp1 WT/KO/TG mice with proven Casp1C284AFlag expression.
These data indicate that even tiny amounts of Casp1C284AFlag induced release of other proinflammatory cytokines and that this might contribute to the proinflammatory phenotype observed in our patients. Baseline expression of enzymatically inactive Casp1C284AFlag may be embryonically lethal in mice since not a single mouse could be generated which expressed the transgene under unstimulated conditions. Hence, a conditional Casp1 C284AFlag knock-in mouse model is being established.
This study was supported by the Federal Ministry of Education and Research (BMBF; Deutsches Netzwerk für Primäre Immundefekte PID-NET) and by EU Marie Curie International Reintegration grant no. GA-2007-224894 (F.P.).
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.