B6.Cg-Gt(ROSA)26Sor^{tm1(CAG-HIST1H2BJ/mCherry,-EGFP/Rpl10a)Evdr}/J

Stock number: 029789
Product name: H2B-TRAP, TRAP , EGFP-L10a
Research areas: Cancer Research, Cell Biology Research, Immunology, Inflammation and Autoimmunity Research, Neurobiology Research, Research Tools

Description

The H2B-TRAP allele (Translating Ribosome Affinity Purification) has a loxP-flanked STOP preventing transcription of two individual components: H2B/mCherry and EGFP/L10a. When expressed, the H2B/mCherry protein allows nuclei-labeling with mCherry (which enables nuclear isolation by fluorescence-based purification). Furthermore, EGFP/L10a fluorescently tags the translating mRNA polysome complex (which enables isolation of RNAs that are actively engaged by ribosomes). Overall, H2B-TRAP 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 NuTRAP (Stock No. 029899).

Detailed description

The H2B-TRAP allele (Translating Ribosome Affinity Purification) has a loxP-flanked STOP preventing transcription of two individual components that are separated by a self-cleaving viral 2A peptide: [i] H2B/mCherry (the human histone 1 H2bj fused to mCherry) and [ii] 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 two components should be prevented by the floxed-STOP cassette. The donating investigator reports they observe no mCherry or EGFP expression prior to the introduction of Cre recombinase. Mice heterozygous for the H2B-TRAP allele are viable and fertile with no reported gross physical or behavioral abnormalities. To date (January 2017), it has not been attempted to make this strain homozygous.

Upon exposure to Cre recombinase, the H2B-TRAP allele co-expresses H2B/mCherry and EGFP/L10a. The H2B/mCherry protein allows nuclei-labeling with mCherry (direct fluorescence) - this enables nuclear isolation by fluorescence-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, H2B-TRAP mice may be bred to have Cre recombinase expression in hepatocytes (via Albumin-Cre ; Stock No. 003574), agouti-related protein-secreting neurons (via Agrp-Ires-cre; see Stock No. 012899) or active populations of neurons in the brain (via Arc^CreERT2 ; Stock No. 021881). The resulting Alb-H2B-TRAP, AgRP-H2B-TRAP or ArcTRAP mice (respectively) showed mCherry and EGFP-labeling in the expected cell types, with no abnormal phenotypes reported.


In addition, when bred to have adipocyte-specific Cre recombinase expression (via Adiponectin-Cre ; Stock No. 010803), the resulting Ad-H2B-TRAP mice show brown adipose tissue (BAT) adipocytes have H2B/mCherry-labeled nuclei and EGFP/L10a-labeled ribosomes in cytoplasm. However, Ad-H2B-TRAP mice were profoundly lipodystrophic in all white adipose tissue (WAT) depots and failed to gain weight on high-fat diet, as compared to similarly treated control animals. As such, the donating investigator does not recommend using H2B-TRAP mice for work in adipocytes - suggesting instead that adipocyte studies may better utilize their NuTRAP mouse line (Stock No. 029899).

Of note, the frt sites flanking the H2B-TRAP 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 H2B-TRAP allele (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::hH2B-mCherry::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 H2B-mCherry fusion protein (coding sequence for the human histone 1 H2bj gene [HIST1H2BJ] fused in-frame to the N-terminus of a monomeric red fluorescent protein sequence [mCherry]), 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 H2B-TRAP colony was bred with C57BL/6J wildtype mice for at least ten generations 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).

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-HIST1H2BJ/mCherry,-EGFP/Rpl10a)Evdr}
Name: targeted mutation 1, Evan Rosen
MGI accession ID: MGI:5817798
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: Rpl10a,ribosomal protein L10A,mouse, laboratory; H2BC11,H2B clustered histone 11,human; RFP,Red Fluorescent Protein,coral; GFP,Green Fluorescent Protein,
Site of expression: Upon exposure to Cre recombinase, the H2B-TRAP allele co-expresses H2B (human histone 1 H2bj)/mCherry and EGFP/L10a fusion proteins.
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, the H2B-mCherry fusion protein (coding sequence for the human histone 1 H2bj gene [HIST1H2BJ] fused in-frame to the N-terminus of a monomeric red fluorescent protein sequence [mCherry]), 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]), WPRE,a polyA signal, an AttB site, 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.