B6;129P2-Gt(ROSA)26Sor^{tm1(CAG-RABVgp4,-TVA)Arenk}/J

Stock number: 024708
Product name: RphiGT
Strain synonyms: RΦGT
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

In 2023, the RΦGT allele in Stock No. 024708 was discovered to have always had a 1bp insertion immediately following the TVA start codon that causes a frameshift and the resulting protein is predicted to be non-functional. The 1bp insertion is not expected to adversely affect the Cre-inducible expression of functional RABVgp4. See Detailed Description section for more information.

When used in conjunction with a mutant pseudotyped (EnvA) rabies virus reporter, Rabies-SADdeltaG-EGFP, and a functional TVA and an appropriate Cre-expressing strain, this RΦGT mouse line may be useful for monosynaptic neuron tracing.

Detailed description

In 2023, the RΦGT allele in Stock No. 024708 was discovered to have always had a 1bp insertion immediately following the TVA start codon that causes a frameshift and the resulting protein is predicted to be non-functional. The 1bp insertion is not expected to adversely affect the Cre-inducible expression of functional RABVgp4. Additional details are below.

These mice have a CAG (CMV enhancer/chicken beta-actin promoter), loxP-flanked STOP fragment, recombinant rabies glycoprotein G gene (RABVgp4), IRES and TVA sequence (see 2023 note below) inserted into the Gt(ROSA)26Sor locus. When used in conjunction with a mutant ASLV-A pseudotyped (EnvA) rabies virus GFP vector, Rabies-SADΔG-EGFP(EnvA)-EGFP, this RΦGT strain may be useful as a reporter strain for monosynaptic neuron tracing. For example, introduction of a functional TVA allows the Rabies-SADΔG-EGFP(EnvA)-EGFP to enter the cell, while expression of the rabies virus glycoprotein allows spreading of the viral particles to other cells with direct synaptic contact. Secondarily infected cells will not express the rabies virus glycoprotein, thus being unable to further spread the Rabies- SADΔG-EGFP(EnvA)-EGFP particle. There is no spreading of the Rabies- SADΔG-EGFP(EnvA)-EGFP without Cre recombinase activity. Mice that are homozygous for the targeted mutation are viable and fertile.

TVA sequence is predicted to be non-functional [2023]:
In 2023, a researcher reported the RΦGT mice express a non-functional TVA after Cre exposure. To investigate, The Jackson Laboratory quality control team sequenced the IRES-TVA region from Stock No. 024708 cryopreserved sperm (frozen from the homozygous male we originally received in 2014) and a cohort of mice from our live colony. Compared to the 360bp TVA cDNA reference sequence that encodes a 120 aa protein (cited in the original publication as pCMMP-TVA800 and the sequence files provided by the donating investigator), our analyses revealed a 1bp guanine insertion immediately following the TVA start codon (the TVA sequence was otherwise the same as the reference sequence). The extra G causes a frameshift and the resulting protein is predicted to be 15 aa before encoding a stop codon (presumably non-functional). This indicates the male we received in 2014 and was used as founder of all of our live colony encodes a frameshifted, non-functional TVA. To date, The Jackson Laboratory has received no reports of any trouble regarding this strain's expression of a functional recombinant rabies glycoprotein G (RABVgp4) after Cre exposure.

Development

To create the RΦGT allele, a targeting vector containing a floxed STOP cassette, recombinant rabies glycoprotein G cDNA, IRES sequence and TVA (avian leukosis and sarcoma virus subgroup A receptor, chicken) cDNA under the control of CAG (CMV enhancer/chicken beta-actin promoter) was inserted into the Gt(ROSA)26Sor locus. See Detailed Description section for more information regarding TVA. The construct was electroporated into 129P2/OlaHsd-derived E14 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. The donating investigator reports the mice were then backcrossed to C57BL/6 for 5 generations (see SNP note below) prior to sending one homozygous male to The Jackson Laboratory Repository in 2014. During backcrossing, the Y chromosome was not fixed to the C57BL/6 genetic background. Upon arrival in 2014, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 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 rederived living colony at The Jackson Laboratory Repository. Three of the 27 markers throughout the genome were segregating, suggesting an incomplete backcross.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-RABVgp4,-TVA)Arenk}
Name: targeted mutation 1, Benjamin Arenkiel
MGI accession ID: MGI:5550559
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inserted expressed sequence
Synonyms: RPhiGT
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: 129P2/OlaHsd
Expressed genes: TVA,subgroup A avian leukosis virus receptor,
Molecular note: The targeting vector contains a loxP-flanked STOP cassette, recombinant rabies glycoprotein G, IRES sequence and TVA (avian leukosis and sarcoma virus subgroup A receptor, chicken) under the control of CAG (CMV enhancer/chicken beta-actin promoter). Expression of the rabies glycoprotein is blocked by the loxP-flanked STOP fragment.