C57BL/6-Tg(Ins2-TFRC/OVAL*)mT4Edp/EhrlJ

Stock number: 039869
Product name: RIP-mT4
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

The RIP-mT4 mice have transgenic RIP (Rat Insulin Promoter; Ins2) driving a modified membrane-bound form of chicken ovalbumin (mOVA), mT4 expression in pancreatic beta cells. This transgenic model has been used to study ectopic expression of tissue-restricted antigens (TRA) by medullary thymic epithelial cells (mTECs) for central tolerance and T cell-mediated autoimmunity. These mice may be useful in autoimmune disease and diabetes research.

Detailed description

The RIP-mT4 mice have transgenic RIP (Rat Insulin Promoter; Ins2) driving a modified membrane-bound form of chicken ovalbumin (OVAL), mT4. The hemizygote transgenic mice are viable, fertile, normal in size, and do not display gross physical or behavioral abnormalities. The mT4 variant of OVA (mOVA) is expressed in pancreatic beta cells and has a threshold affinity for the OT-I TCR (OVA transgenic-I T-cell receptor). When RIP-mT4 is mated to C57BL/6-Tg(TcraTcrb)1100Mjb/J (commonly referred to as OT-1 and recognizes OVA-specific T-cells, Stock No. 003831), the blood glucose level of newborn mice was elevated and high mortality within the first 2 wk after birth was observed in double transgenic mice. This transgenic model has been used to study ectopic expression in medullary thymic epithelial cells of moderate affinity antigens for the OT-I TCR to assess the requirements for central tolerance.

Development

The fusion transgene mT4 is under the control of the rat insulin promoter (Ins2) and encodes the first 118 residues of the human transferrin receptor (TFRC), which includes the cytoplasmic tail and signal/anchor domain, fused to mature chicken ovalbumin (OVAL), residues 139-387 coding for (SIINFEKL), resulting in a membrane-bound form of ovalbumin. The cDNA of the transferrin-receptor-OVA protein (TFRC/OVA) construct was cloned into the pBlue Vector. Then, site-directed mutagenesis was used to generate RIP-mT4 (SIITFEKL) cDNAs. A 3.2 kB fragment was injected into the pronucleus of fertilized C57BL/6 zygotes and transplanted into foster mothers. Founder mice were obtained, and one founder line (mT4) was bred to C57BL/6 genetic background mice and maintained by Dr. Ed Palmer's lab at the University of Basel. The copy numbers and insertion site of the transgene are unknown. Frozen embryos of RIP-mT4 were obtained by Dr. Lauren Ehrlich in 2016 and were rederived in 2024 from embryo transfer at UT Austin. A RIP-mT4+ male was then mated to C57BL/6J (Stock No. 000664) females for a single generation by the donating laboratory. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

In 2025, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. One of the 43 markers throughout the genome was segregating with an unknown source. Also, 4 of the 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

In 2025, the one recovered carrier male from the original males sent to The Jackson Laboratory Repository also showed variable for L253F and E254T [the small region 20-25 nt upstream of the SIITFEKL region], suggesting multiple variable transgene copies at the insertion site. No phenotypic consequence of these variations is assessed at this time.

Allele

Symbol: Tg(Ins2-TFRC/OVAL*)mT4Edp
Name: transgene insertion mT4, Ed Palmer
MGI accession ID: MGI:7858863
Generation method: Transgenic
Attributes: Inserted expressed sequence
Synonyms: RIP-mT4
Marker symbol: Tg(Ins2-TFRC/OVAL*)mT4Edp
Marker name: transgene insertion mT4, Ed Palmer
Chromosome: UN
Strain of origin: C57BL/6
Molecular note: The transgene consists of the rat insulin promoter (Ins2) driving the first 118 residues of the human transferrin receptor (TFRC), which includes the cytoplasmic tail and signal/anchor domain, fused to mature chicken ovalbumin (OVAL), residues 139-387 coding for (SIINFEKL), which was mutated by site-directed mutagenesis to (SIITFEKL) resulting in a membrane-bound form of ovalbumin.