C57BL/6-Igs2^{tm1(CAG-TurboID)Long}/J

Stock number: 041107
Product name: LSL-ERTurboID
Research areas: Research Tools

Description

LSL-ERTurboID knock-in mice have Cre-inducible expression of an HA- and V5-tagged endoplasmic reticulum-directed biotin ligase sequence, ERTurboID. Cre-mediated recombination results in the excision of a floxed STOP cassette, enabling the tissue and time-dependent expression of ERTurboID. This is a tool for the isolation and purification of proteins and protein complexes trafficking through the secretory pathway upon biotin administration.

Detailed description

LSL-ERTurboID knock-in mice have CAG promoter directing Cre-inducible expression of an HA- and V5-tagged endoplasmic reticulum-directed biotin ligase sequence, ERTurboID, inserted into the Igs2 locus. Cre-mediated recombination results in the excision of a floxed STOP cassette, enabling the tissue and time-dependent expression of ERTurboID.

In these LSL-ERTurboID, a floxed STOP cassette blocks downstream expression of the ERTurboID cassette. After breeding to a Cre-expressing line, these mice secrete biotinylated proteins in a tissue-specific fashion when given exogenous biotin in their diet. This allows highly specific biotinylation and purification of proteins trafficking through the secretory pathway and permits the analysis of cellular secretomes through streptavidin affinity purification and quantitative mass spectrometry proteomics.

Homozygous mice are viable and fertile.

Development

The LSL-ERTurboID targeting vector was created using the Igs2 TARGATT site-specific ΦC31 integrase-mediated cassette exchange to contain, from 5' to 3', a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a floxed STOP cassette containing 3 SV40 late polyA signals, an HA- and V5-tagged biotin ligase sequence fused to an endoplasmic reticulum-targeting Igκ leader sequence and an endoplasmic reticulum retention signal (RDEL). The donor plasmid also contained an ampicillin resistance (ampR) cassette. This targeting vector was inserted into the Igs2 locus. The construct was microinjected, along with the ΦC31 integrase mRNA, into the pronuclei of zygotes collected from TARGATT B6J/H11-attP3 mice on a C57BL/6J genetic background, which were then implanted into recipient pseudo-pregnant females. TARGATT H11-attP3 mice were generated by knocking three attP sites into the Igs2 locus. Subsequent progeny were screened by PCR for the presence of the LSL-ERTurboID allele. The att-flanked ampR was removed by ΦC31. These mice were further backcrossed to C57BL/6J mice (Stock No. 000664) for at least 6 generations by the donating laboratory (see SNP note below). Cryopreserved sperm was sent to The Jackson Laboratory. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

In 2026, genotyping at The Jackson Laboratory identified the presence of an ampicillin resistance gene (AMP) that is segregating with LSL-ERTurboID allele. Also, 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 rederived living colony at The Jackson Laboratory Repository. One of the 43 markers throughout the genome was segregating with an unknown source on Chromosome 11 which is likely linked to the mutant allele. Also, 2 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.

Allele

Symbol: Igs2^{tm1(CAG-TurboID)Long}
Name: Jonathan Long
MGI accession ID: MGI:8246684
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Epitope tag
Synonyms: LSL-ERTurboID
Marker symbol: Igs2
Marker name: intergenic site 2
Marker synonyms: H11; Hipp11; Iis2
Chromosome: 11
Strain of origin: C57BL/6J
Molecular note: Site-specific PhiC31 integrase-mediated transgenesis is used to insert an LSL-ERTurboID into the intergenic site 2 locus (Hipp11). The targeting vector used for the Igs2 TARGATT site-specific Phi C31 integrase-mediated cassette exchange consists of from 5' to 3', a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a floxed STOP cassette containing 3 SV40 late polyA signals, an HA- and V5-tagged biotin ligase sequence fused to an endoplasmic reticulum-targeting Igk leader sequence, and an endoplasmic reticulum retention signal (RDEL). The construct was microinjected, along with the PhiC31 integrase mRNA, into the pronuclei of zygotes collected from TARGATT B6J/H11-attP3 mice on a C57BL/6J genetic background.