• TWEAK is a 27 kDa TNF superfamily protein (TNFSF12) existing as a membrane-bound or soluble transmembrane protein; By binding its receptor FN14, it induces apoptosis, activates NF-κB, promotes angiogenesis and endothelial cell proliferation, and triggers inflammatory cytokines.
• APRIL, a 32 kDa TNF ligand superfamily member (TNFSF13), is proteolytically processed by furin into a 17 kDa soluble molecule that signals through TACI/BCMA receptors to promote T/B-cell proliferation, induces IgA class switching, inhibits apoptosis, and accumulates in cancers via tumor-infiltrating neutrophil expression. TWE-PRIL is a bioactive chimeric protein comprising TWEAK’s intracellular, transmembrane, and stalk domains fused to APRIL’s TNF homology domain, expressed on monocytes and activated T cells with stable cell-surface presentation.
• The entire genome encoding rat TWEAK gene and April gene was replaced by human counterparts of TWEAK and APRIL, respectively.
• Rat TWEAK and April mRNA were only detectable in wild-type SD rats. Human TWEAK and APRIL mRNA were exclusively detectable in homozygous B-hTWEAK/hAPRIL rats. TWEAK was detected wild-type SD rats and homozygous B-hTWEAK/hAPRIL rats due to the cross-reactivity of antibody. Soluble human APRIL was exclusively detectable in homozygous B-hTWEAK/hAPRIL rats but not in wild-type SD rats.
• This product is used for pharmacological and safety evaluation of autoimmune diseases such as psoriasis and IgA nephropathy.

Gene targeting strategy for B-hTWEAK/hAPRIL rats.

The genomic region of rat Tweak gene that encodes the full-length protein was replaced by human counterparts in B-hTWEAK/hAPRIL rats. The promoter and 5’UTR region of the rat gene were retained. The 3’UTR region of the rat gene was replaced by human counterparts. The human TWEAK expression was driven by endogenous rat Tweak promoter, while rat Tweak gene transcription and translation will be disrupted.

The genomic region of rat April gene that encodes the full-length protein was replaced by human counterparts in B-hTWEAK/hAPRIL rats. The promoter, 5’UTR region and 3’UTR region of the rat gene was replaced by human counterparts. The human APRIL expression was driven by endogenous rat April promoter, while rat April gene transcription and translation will be disrupted.

Strain specific analysis of TWEAK and APRIL mRNA expression in wild-type SD rats and B-hTWEAK/hAPRIL rats by RT-PCR. Lung and liver RNA were isolated from wild-type SD rats (+/+) and homozygous B-hTWEAK/hAPRIL rats (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with rat or human TWEAK and APRIL primers. Rat TWEAK and April mRNA were only detectable in wild-type SD rats. Human TWEAK and APRIL mRNA were exclusively detectable in homozygous B-hTWEAK/hAPRIL rats but not in wild-type SD rats.

Western blot analysis of TWEAK (TNFSF12) protein expression in homozygous B-hTWEAK/hAPRIL rats. Various tissue lysates were collected from wild-type SD rats (+/+) and homozygous B-hTWEAK/hAPRIL rats (H/H), and then analyzed by western blot with anti-TWEAK antibody (proteintech, 12537-1-AP). TWEAK was detected in spleen, liver, kidney, lung, colon and heart from wild-type SD rats and homozygous B-hTWEAK/hAPRIL rats due to the cross-reactivity of antibody.

Strain specific APRIL expression analysis in wild-type SD rats and homozygous humanized B-hTWEAK/hAPRIL rats by ELISA. Serum was collected from wild-type SD rats (female, n=3, 14-week-old) and homozygous B-hTWEAK/hAPRIL rats (female, n=3, 14-week-old). Expression levels of human APRIL were analyzed by ELISA (anti-human APRIL ELISA Kit: invitrogen, BMS2008). Human APRIL was exclusively detectable in homozygous B-hTWEAK/hAPRIL rats, but not in wild-type SD rats. Values are expressed as mean ± SEM. ND: not detectable.