C57BL/6N-Trac^em2Gmrs/J

Stock number: 039124
Product name: B6.TCRA-RFP
Research areas: Research Tools

Description

B6.TCRA-RFP knock-in mice contain an insertion of tdTomato at the C-terminal of the T cell receptor alpha constant (Trac) gene. These mice, together with B6.TCRA-GFP mice (Stock No. 039123), enable evaluation of the entire repertoire of TCRα expression by flow cytometry and confocal microscopy, removing the limitation of anti-TCRVα mAb labeling and providing the ability to distinguish T cells expressing a single TCRα from those coexpressing two TCRα chains.

Detailed description

B6.TCRA-RFP knock-in mice contain a CRISPR/Cas9-generated insertion of tdTomato at the 3’ end of exon 3, at the C-terminal end of the T cell receptor alpha constant (Trac) gene attached by an 18-amino-acid flexible linker protein. The RFP insertion does not affect endogenous gene expression and does not interfere with TCR trafficking or function. In these B6.TCRA-RFP knock-in mice, expression of RFP is specific for CD4⁺ and CD8⁺ T cells. Confocal microscopy confirms that RFP localizes to the T cell membrane. RFP is first detectable at low levels in double-positive CD4⁺CD8⁺ (DP) thymocytes and increases in expression throughout maturation to single-positive CD4⁺CD8^- (CD4SP) and CD4^-CD8⁺ (CD8SP) thymocytes, consistent with increasing TCRα expression during thymopoiesis. B6.TCRA-RFP mice have normal thymic development, with equivalent generation of CD4SP and CD8SP thymocytes at frequencies similar to wild-type B6 mice. Reporter mice have normal frequencies of CD4⁺ and CD8⁺ T cells in the spleen. CD4⁺ and CD8⁺ T cells from reporter mice proliferated equivalently to T cells from wildtype B6 mice following in vitro stimulation with plate-bound anti-CD3 and anti-CD28, indicating that GFP does not interfere with TCR function.

Fluorescence is detectable by direct microscopy and flow cytometry. Homozygous mice are viable and fertile.

Most (>90%) dual T cell receptor (TCR) cells result from production of two functional TCRα proteins, each paring with the cell’s single TCRβ. Interbreeding of these B6.TCRA-RFP knock-in mice to B6.TCRA-GFP knock-in mice (Stock No. 039123) produces mice with one TRAC chromosomal copy containing the RFP reporter and the other containing the GFP reporter (B6.TCRA-GFP/RFP). This provides the ability to distinguish T cells expressing a single GFP or RFP TCRα from those coexpressing two TCRα chains. Used together, B6.TCRA-GFP and B6.TCRA-RFP knock-in mice can be used to overcome the limitations of existing approaches for identification of single- and dual-TCR cells.

Development

A Guide RNA (CTGAGGCTGTGGTCCAG/TTG(AGG)) was designed to insert a red fluorescent protein (tdTomato) sequence at the 3’ end of exon 3, at the C-terminal end, of the T cell receptor alpha constant (Trac) gene attached by an 18-amino-acid flexible linker protein (GSGSGSGSGSGSGSQPVAT). The guide RNA, ssDNA donor plasmid, and the cas9 nuclease were electroporated into the cytoplasm of C57BL/6NCrl-derived zygotes with well recognized pronuclei. Targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by flow cytometry of peripheral blood lymphocytes and sequencing and resulting B6.TCRA-RFP mice were bred to C57BL/6NCrl mice for at least 2 generations to limit the presence off-target CRISPR changes. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304).

Allele

Symbol: Trac^em2Gmrs
Name: endonuclease-mediated mutation 2, Gerald P Morris
MGI accession ID: MGI:7576105
Generation method: Endonuclease-mediated
Attributes: Reporter
Synonyms: TCRA-RFP
Marker symbol: Trac
Marker name: T cell receptor alpha constant
Marker synonyms: Gm16914; IMD7; TCRA; Tcra-C; TRA@; TRCA
Chromosome: 14
Strain of origin: C57BL/6NCrl
Molecular note: Using CRISPR/Cas9 technology, a red fluorescent protein (tdTomato) sequence at the 3' end of exon 3, at the C-terminal end of the T cell receptor alpha constant (Trac) gene attached by an 18-amino-acid flexible linker protein (GSGSGSGSGSGSGSQPVAT).