B6;129S6-Gt(ROSA)26Sor^{tm2(CAG-NuTRAP)Evdr}/J

Stock number: 029899
Product name: NuTRAP , RANGAP-TRAP
Research areas: Cancer Research, Cell Biology Research, Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

The NuTRAP allele (Nuclear tagging and Translating Ribosome Affinity Purification) has a loxP-flanked STOP preventing transcription of three individual components: BirA, BLRP-tagged mCherry/mRANGAP1 and EGFP/L10a. When expressed, the BLRP-tagged mCherry/mRANGAP1 protein is biotinylated by BirA, allowing nuclear membrane-labeling with mCherry and biotin (which enables nuclear isolation by fluorescence- and affinity-based purification). Furthermore, EGFP/L10a fluorescently tags the translating mRNA polysome complex (which enables isolation of RNAs that are actively engaged by ribosomes). Overall, NuTRAP mice are a Cre-inducible tool strain that allows labeling and simultaneous isolation of cell type-specific nuclei and mRNA, and are well-suited for studying epigenomics and transcriptomics from specific cell types within a heterogeneous tissue. Of note, the donating investigator also has available their similar mouse line H2B-TRAP (Stock No. 029789).

Detailed description

The NuTRAP allele (Nuclear tagging and Translating Ribosome Affinity Purification) has a loxP-flanked STOP preventing transcription of three individual components each separated by a self-cleaving viral 2A peptide: [i] BirA (the E. Coli biotin ligase), [ii] BLRP-tagged mCherry/mRANGAP1 (the mouse nuclear membrane RAN GTPase activating protein 1 fused to mCherry and tagged with a biotin ligase recognition peptide) and [iii] EGFP/L10a (the 60S ribosomal subunit L10a fused to EGFP).

Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of the three components should be prevented by the floxed-STOP cassette. The donating investigator reports they observe no BirA, mCherry or EGFP expression prior to the introduction of Cre recombinase. Mice homozygous for the NuTRAP allele are viable and fertile with no reported gross physical or behavioral abnormalities.

Upon exposure to Cre recombinase, the NuTRAP allele co-expresses BirA, BLRP-tagged mCherry/mRANGAP1 and EGFP/L10a. The BLRP-tagged mCherry/mRANGAP1 protein is biotinylated by BirA, allowing nuclear membrane-labeling with mCherry (direct fluorescence) and biotin - this enables nuclear isolation by two independent methods: fluorescence- and affinity-based purification. Furthermore, EGFP/L10a fluorescently tags the translating mRNA polysome complex (direct fluorescence in the cytoplasm and nucleolus) - this enables isolation of RNAs that are actively engaged by ribosomes.
Specifically, when bred to have adipocyte-specific Cre recombinase expression (via Adiponectin-Cre ; Stock No. 010803), the resulting Ad-NuTRAP mice showed EGFP/L10a in brown adipose tissue (BAT) and white adipose tissue (WAT), but not in kidney or liver. Fluorescence microscopy revealed mCherry/mRANGAP1 on the nuclear surface of lipid-filled adipocytes and EGFP/L10a in the cytoplasm and nucleolus. Immunohistological analysis using anti-GFP staining showed that adipocytes were specifically labeled.
When bred to have hepatocyte-specific Cre recombinase expression (via Albumin-Cre ; Stock No. 003574), the resulting Alb-NuTRAP mice showed EGFP-labeled hepatocytes, and mCherry-positive hepatocyte nuclei were able to be isolated by flow cytometry.

Of note, the frt sites flanking the NuTRAP allele allow for additional targeted replacement of the reporter sequences through FLP-mediated recombination, if so desired. Also, the AttB/AttP-flanked selection cassette may be removed by introduction of the site-specific bacteriophage PhiC31 integrase, if so desired.

Development

The NuTRAP allele (Nuclear tagging and Translating Ribosome Affinity Purification) was created in the laboratory of Dr. Evan D. Rosen (Beth Israel Deaconess Medical Center and Harvard Medical School).


The Rosa26::pCAG::LSL::BirA::2A::BLRP-mCherry-mRANGAP1::2A::EGFP-L10a::WPRE::bGHpA targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a frt site, a loxP-flanked STOP cassette (with stop codons in all three reading frames linked to three repeats of the SV40 polyA signal), the E. Coli biotin ligase gene (BirA), a self-cleaving viral 2A peptide, the BLRP-mCherry-mRANGAP1 fusion gene (described below), a self-cleaving viral 2A peptide, the EGFP-L10a fusion protein (an enhanced green fluorescent protein gene [EGFP] fused in-frame to the N-terminus of a cDNA sequence encoding the mouse 60S ribosomal subunit L10a [Rpl10a]), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal (bGHpA), an AttB site, a Neo cassette (PGK promoter-frt-Neomycin resistance gene-PGK polyA), and an AttP site. This entire targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were selected. Chimeric mice were bred with C57BL/6J mice to obtain germ-line transmission. The donating lab reported that the resulting NuTRAP colony was bred with C57BL/6J wildtype mice for at least ten generations (see SNP note below) prior to sending males with black coat color to The Jackson Laboratory Repository in 2017. Upon arrival, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

In 2017, 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 and our first generation rederived living colony. Both cohorts showed all markers throughout the genome were C57BL/6 allele-type with the exception of three markers segregating for 129S allele-type markers (one on chromosome 10 [~60 Mbp], 12 [~90 Mbp] and X [~70 Mbp]). Importantly, all 5 markers that determine C57BL/6J from C57BL/6N were found to be C57BL/6J allele-type. Collectively, these data suggest that the mice sent to The Jackson Laboratory Repository were ~93-94% C57BL/6.

The BLRP-mCherry-mRANGAP1 fusion gene consists of a monomeric red fluorescent protein sequence (mCherry) fused in-frame to the N-terminal of a cDNA sequence encoding the mouse nuclear membrane RAN GTPase activating protein 1 [Rangap1 ; mRANGAP1]. This fusion gene is tagged at the mCherry N-terminal with a biotin ligase recognition peptide (BLRP ; a 23 amino acid protein with AviTag core [MAGGLNDIFEAQKIEWHEDTGGS]). Enzymatic biotinylation with E. coli biotin ligase (BirA) is highly specific in covalently attaching biotin to the 15 amino acid AviTag peptide, giving a homogeneous product with high yield.

Allele

Symbol: Gt(ROSA)26Sor^{tm2(CAG-NuTRAP)Evdr}
Name: targeted mutation 2, Evan Rosen
MGI accession ID: MGI:5817820
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: RFP,Red Fluorescent Protein,coral; GFP,Green Fluorescent Protein,
Site of expression: Upon exposure to Cre recombinase, the NuTRAP allele co-expresses BirA (E. Coli biotin ligase), BLRP (biotin ligase recognition peptide)-tagged mCherry/mRANGAP1 (the mouse nuclear membrane RAN GTPase activating protein) and EGFP/L10a (the 60S ribosomal subunit L10a) fusion proteins in cre expressing tissues.
Molecular note: The targeting vector was designed with (from 5' to 3') with a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter, a FRT site, a loxP-flanked STOP cassette, the E. Coli biotin ligase gene (birA), a 2A peptide, the BLRP-mCherry-mRANGAP1 fusion gene (described below), a 2A peptide, the EGFP-L10a fusion protein (an enhanced green fluorescent protein gene [EGFP] fused in-frame to the N-terminus of a cDNA sequence encoding the mouse 60S ribosomal subunit L10a [Rpl10a]), a WPRE, a polyA signal, an AttB site, a PGK-FRT-Neo-PGK polyA, and an AttP site. This entire targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. The BLRP-mCherry-mRANGAP1 fusion gene consists of a monomeric red fluorescent protein sequence fused in-frame to the N-terminal of a cDNA sequence encoding the mouse nuclear membrane RAN GTPase activating protein 1. This fusion gene is tagged at the mCherry N-terminal with a biotin ligase recognition peptide (BLRP ; a 23 amino acid protein with AviTag core.