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Targeting strategy 1. For conditional knockout, the loxP fragments are usually inserted into the introns downstream of the ATG-containing-exon and removal of the flanked exon(s) will result in frame-shift for protein translation. 2. This design is to conditionally knockout the exon 2 and exon3 by Cre-loxP system. The intron 1 is big and insertion of 5’ loxP element will not interfere ier3ip1 mRNA splicing. 3’loxP will be inserted into intergenic region downstream of ier3ip1 and upstream of the promoter-assoicated elements of Hdhd2.

Targeting strategy1. For conditional knockout, the loxP fragments are usually inserted into the introns downstream of the ATG-containing-exon and removal of the flanked exon(s) willresult in frame-shift for protein translation. After scanning the gene structure and the size of exons, we found that exon 2 (ENSMUSE00001271873) and exon3 (ENSMUSE00001293257) can be conditionally removed and will result in 30 aa (Residual N-terminal, unknown C-terminal ) truncated protein.2. This design is to conditionally knockout the exon 2 and exon3 by Cre-loxP system. The intron 1 is big and insertion of 5’ loxP element will not interfere ier3ip1 mRNAsplicing. 3’loxP will be inserted into intergenic region downstream of ier3ip1 and upstream of the promoter-assoicated elements of Hdhd2. To minimize the possibility of disruption of mRNA splicing , 3’ loxP site will be inserted into the nonconserved region .