B6;129S-Gt(ROSA)26Sor^{tm1.1(CAG-TVA,-EGFP,-RABVgp4*)Velm}/J

Stock number: 041408
Product name: LSL-TVAoG , Rosa26:LSL-TVA-P2A-EGFP-P2A-oG
Research areas: Neurobiology Research, Research Tools

Description

TVAoG (Rosa26:LSL-TVA-P2A-EGFP-P2A-oG) mice enable Cre-inducible, optimized monosynaptic rabies tracing of neuronal microcircuits at single-cell resolution.

Detailed description

Monosynaptic rabies virus (mRV) tracing has become a cornerstone technology for dissecting neuronal circuits, enabling precise identification of first-order presynaptic partners of defined neurons.

TVAoG (Rosa26:LSL-TVA-P2A-EGFP-P2A-oG) mice carry a Gt(ROSA)26Sor knock-in allele in which a CAG promoter and loxP-flanked Stop cassette precede avian-specific retroviral receptor (TVA), enhanced green fluorescent protein (EGFP), and optimized rabies glycoprotein (oG) sequences. After Cre recombinase-mediated recombination, the Stop cassette is removed, enabling expression of TVA, the receptor required for infection by EnvA-pseudotyped rabies virus, as well as oG, the optimized rabies glycoprotein that supports trans-synaptic spread. The construct also includes EGFP as a fluorescent reporter, allowing visualization of cells in which the allele is activated. The donating laboratory notes that EGFP fluorescence is readily detectable by FACS, but is only weakly visible in fresh tissue sections. The signal improves after immunofluorescent amplification. Heterozygotes and homozygotes are viable, fertile, and show no overt phenotype.

The donating laboratory reports that TVAoG mice show markedly improved infection efficiency and broader starter-cell labeling than the RΦGT model (Stock No. 024708) across diverse neuronal types. By combining this system with barcoded rabies libraries and subcellular resolution unbiased spatial transcriptomics, high-throughput, spatially resolved connectome reconstruction can be achieved.

Development

A CAG-loxP-Stop-loxP-TVA-P2A-EGFP-P2A-oG-attB-neomycin-attP cassette was inserted into the mouse Gt(ROSA)26Sor locus via homologous recombination in (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. CAG is the CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter. TVA represents an avian-specific retroviral receptor required for infection by EnvA-pseudotyped rabies virus. EGFP represents the enhanced green fluorescent reporter. oG is the optimized rabies glycoprotein that supports trans-synaptic spread (derived from pCAG-oG, Addgene #74288; PMID: 27149846). Resultant mice were crossed to C57BL/6J mice (Stock No. 000664) to confirm germline transmission, then with the phiC31 strain (Stock No. 007670) to excise the attB/attP-flanked neomycin selection cassette. Founders were crossed with C57BL/6J mice for one more generation to remove phiC31. Upon arrival, sperm was cryopreserved. To establish a live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Gt(ROSA)26Sor^{tm1.1(CAG-TVA,-EGFP,-RABVgp4*)Velm}
Name: targeted mutation 1.1, Dmitry Velmeshev
MGI accession ID: MGI:8275289
Generation method: Targeted
Attributes: Reporter
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: A CAG-loxP-Stop-loxP-TVA-P2A-EGFP-P2A-oG-attB-neomycin-attP cassette was inserted into the mouse Gt(ROSA)26Sor locus via homologous recombination. CAG is the CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter. TVA represents an avian-specific retroviral receptor required for infection by EnvA-pseudotyped rabies virus. EGFP represents the enhanced green fluorescent reporter. oG is the optimized rabies glycoprotein that supports trans-synaptic spread. The neo cassette was removed with the phiC31 recombinase.