The RiboTag targeted mutation of the ribosomal protein L22 locus allows Cre-mediated hemagglutinin epitope tagging of ribosomes from user-defined cell types and/or immunoprecipitation of ribosomes bound to mRNA from those specific cell types.
Of note, this Rpl22 floxed allele is also available on a C57BL/6J genetic background as Stock No. 029977, without the retinal degeneration 8 mutation.
Paul S Amieux, University of Washington
Mice homozygous for this RiboTag allele are viable and fertile, with a targeted mutation of the ribosomal protein L22 (Rpl22) locus harboring a loxP-flanked wildtype C-terminal exon 4 followed by an identical C-terminal exon 4 that is tagged with three copies of the hemagglutinin (HA) epitope before the stop codon. Prior to exposure to Cre recombinase, RiboTag mice express the wildtype RPL22 protein (15 kDa). When the RiboTag mice are bred to cre-expressing mice, offspring will have the floxed wildtype exon 4 deleted in the cre-expressing tissue and subsequent use of the downstream HA epitope-tagged exon 4 as the terminal exon. The 23 kDa HA epitope-tagged ribosomal protein (RPL22HA) is incorporated into actively translating polyribosomes. These RiboTag mice allow Cre-mediated HA epitope tagging of ribosomes from user-defined cell types and/or immunoprecipitation of ribosomes bound to mRNA from those specific cell types.
A targeting vector was designed to flank the wildtype Rpl22 exon 4 with loxP sites and also place downstream an FRT-flanked neomycin resistance cassette followed by a duplicate C-terminal Rpl22 exon 4 containing three consecutive hemagglutinin epitopes inserted before the Rpl22 stop codon. The donating investigator reports that this construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl+ derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and chimeric males were bred with C57BL/6NCrl females to establish the colony. To remove the FRT-flanked neomycin resistance cassette, mice were bred with FLPeR mice (on a C57BL/6 congenic background; like Stock No. 009086). The resulting RiboTag mice were subsequently backcrossed to C57BL/6NCrl for eight generations prior to arrival at The Jackson Laboratory. Upon arrival, mice were bred to C57BL/6NJ (Stock No. 005304) for at least one generation to establish the colony.
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. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 5 of 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 C57BL/6N genetic background.
|Allele Name||targeted mutation 1.1, Paul S Amieux|
|Allele Type||Targeted (Conditional ready (e.g. floxed), No functional change, Epitope tag)|
|Allele Synonym(s)||RiboTag; Rpl22HA|
|Gene Symbol and Name||Rpl22, ribosomal protein L22|
|Strain of Origin||(129X1/SvJ x 129S1/Sv)F1-Kitl+|
|Molecular Note||A loxP site was inserted upstream of the final coding exon (exon 4) and a loxP site and a frt-flanked neomycin cassette were inserted downstream of the exon. In addition, a modified version of exon 4 was knocked-in downstream of the neo cassette. This duplicated exon 4 contained 3 sequence repeats encoding hemagglutinin eptiope (HA) just upstream of the endogenous stop codon. Founder mice were crossed with FlpeR deleter mice to remove the neomycin cassette leaving exon 4 floxed. In the absence of cre recombinase, the stop codon in the endogenous exon 4 prevents translation of the knocked-in exon and only wild-type protein is expressed. In the presence of cre recombinase, the endogenous exon 4 is removed and the modified exon 4 is instead transcribed leading to a ribosomal complex tagged with HA-epitope.|
|Mutations Made By|| |
Paul Amieux, University of Washington
When maintaining a live colony, homozygous mice may be bred together.
When using the RiboTag mouse strain in a publication, please cite the originating article(s) and include JAX stock #011029 in your Materials and Methods section.