B6.Cg-Igs7^{tm1(tetO-mCardinal,-L_RABVS)Wick}/J

Stock number: 036974
Product name: TRE-CB, TRE-tight-mCardinal-P2A-B19L
Research areas: Neurobiology Research, Research Tools

Description

TRE-CB (TRE-tight-mCardinal-P2A-B19L) mutant mice carry an Igs7 (TIGRE)-targeted mutation that is part of an improved, second-generation monosynaptic tracing system that, unlike previous versions, exerts minimal toxicity. Fluorescent reporter mCardinal and glycoprotein-deleted (ΔG) SAD-B19 rabies virus polymerase variant B19L expression is directed from the TRE-Tight (tetO) promoter when tTA (tetracycline-controlled transactivator) is present.

Detailed description

Monosynatpic tracing is a neural mapping technique that involves the gated replication and spread of a deletion-mutant rabies virus to label neurons directly connected to a targeted population of starting neurons.

TRE-CB (TRE-tight-mCardinal-P2A-B19L) mutant mice carry an Igs7 (TIGRE)-targeted mutation that is part of an improved, second-generation monosynaptic tracing system that, unlike previous versions, exerts minimal toxicity. Fluorescent reporter mCardinal and glycoprotein-deleted (ΔG) SAD-B19 rabies virus polymerase variant B19L expression is directed from the TRE-Tight (tetO) promoter when tTA (tetracycline-controlled transactivator) is present.

The experimental model is produced by introducing a viral vector expressing tTA (to create a Tet-Off system) to triple heterozygous mice expressing any Cre recombinase x TRE-CB x Cre reporter (e.g. Ai14, Stock No. 007914), or any Flp recombinase x TRE-CB x Flp reporter (e.g. Ai65F, Stock No. 032864). Double heterozygous mice expressing TRE-CB x Cre or Flp reporter may also be used in conjunction with a second-generation rabies virus vector incorporating both tTA and Cre or Flp recombinase.

Development

The genes for mCardinal (a basic far red fluorescent protein) and glycoprotein-deleted (ΔG) SAD-B19 rabies virus polymerase variant B19L, separated by a picornavirus 2A element (P2A), were cloned into “Ai62(TITL-tdT) Flp-in replacement vector” (Addgene # 61576) to make the Flp-in construct “TRE-mCardinal-P2A-B19L Flp-in vector” (Addgene #178519). The lox-stop-lox sequence present in the original vector was removed to enable a tetracyline response element (TRE-Tight) to drive mCardinal-P2A-B19L directly with no dependence on Cre recombination. The plasmid was then used for FLP-mediated targeted insertion into the Igs7 locus (also called TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated) in Ai99 Flp-in embryonic stem (ES) cells derived from (129S6/SvEvTac x C57BL/6)F1 background G4 ES cells. Resultant mice were backcrossed to C57BL/6J for more than 10 generations by the donating laboratory (see SNP note below).

Upon arrival at The Jackson Laboratory, sperm was cryopreserved. Cryorecovery/live colony development will involve C57BL/6J (Stock No. 000664).

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. Four of the 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating, including the rd8 recessive mutation associated with retinal degeneration on Chromosome 1 within the Crb1 locus. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Igs7^{tm1(tetO-mCardinal,-L_RABVS)Wick}
Name: targeted mutation 1, Ian Wickersham
MGI accession ID: MGI:6832819
Generation method: Targeted
Attributes: Reporter; Inducible
Synonyms: TRE-CB; TRE-tight-mCardinal-P2A-B19L
Marker symbol: Igs7
Marker name: intergenic site 7
Marker synonyms: Iis7; TIGRE
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: The targeting construct contains a tetracycline-responsive element (TRE; tetO) directing expression of an mCardinal (a basic far red fluorescent protein) and glycoprotein-deleted SAD-B19 rabies virus large structural protein RNA-directed RNA polymerase GTP phosphohydrolase (L_RABVS) variant B19L, separated by a picornavirus 2A element (P2A). The plasmid was then used for FLP-mediated targeted insertion into the Igs7 locus in Ai99 Flp-in embryonic stem (ES) cells derived from (129S6/SvEvTac x C57BL/6)F1 background G4 ES cells.