Rational Design and Construction of a Knock-Out Vector for Targeting the Decoy Type 2 Interleukin -1 Receptor

Given the limited data describing the phenotype for type 2 interleukin -1 receptor (Il1r2-/-) in mouse strains and based on the decoy role of the gene product (IL-1R2), we hypothesized that IL-1R2 may have a unique but similar inhibitory role to IL-1R antagonist (IL-1Ra) in vivo. Despite the anti-inflammatory function of IL-1R2, its role in disease in vivo remains unclear. Therefore, we designed and implemented a knock-out construct template for a mouse embryonic stem (ES) cell line that can be used to easily make knockout mice. Bacterial artificial chromosome (BAC) clone of Il1r2 from mouse strain AB2.2 and ES cells from same strain were obtained and a long chain PCR was performed to isolate homologous arms containing homologous segments (8kb and 2kb). Short segments were cloned out for use as probe sequences and a construct with deletion in exon 3 was made so it can be removed in vivo. The NeoR in cloning vector was then flanked with loxP elements. The two homologous arms were successfully amplified with a truncation in exon 3 of the gene and the wild type cloning plasmid (p1049) was serially modified with loxP elements. One of the successfully transformed plasmid DNA was used as the starting material for the ligation of the subsequent loxP-PacI linker. This plasmid (p1049XL) was amplified in E. coli DH5α cells and DNA extracted. The loxP-PacI linker was successfully ligated into the plasmid and transformed clones screened with MspI and compared to the virtual digest of the theoretical plasmid containing the insert and subsequently sequenced. This study has provided the basic ingredients for making an Il1r2-deficient mouse in order to adequately characterize the phenotype. By assembling the complete knock out construct from templates already provided in this study for the knock out in embryonic stem cells, Il1r2-deficient mice could be made