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.
