The Tlr5fl floxed allele has loxP sites flanking exon 4 of the toll-like receptor 5 gene. Cre recombinase-mediated removal of the floxed sequence creates a null allele. These mice may be useful in studying TLR5 recognition of pathogens (bacterial flagellin), cytokine secretion and activation of innate immune responses.
Of note, a C57BL/6-congenic TLR5 null allele (TLR5-) is also available as Stock No. 028909.
Andrew T Gewirtz, Georgia State University
Benoit Chassaing, Georgia State University
The Tlr5 gene encodes toll-like receptor 5, which has a fundamental role in bacteria recognition and activation of innate immune responses. TLR5 is the receptor for bacterial flagellin, and then acts via MYD88 and TRAF6; leading to NF-kappa-B activation, cytokine secretion and the inflammatory response.
The Tlr5fl allele has loxP sites flanking exon 4 of the Tlr5 gene. Mice homozygous for the Tlr5fl allele are viable and fertile with no reported abnormalities. When bred to mice that express Cre recombinase, the resulting offspring will have absence of Tlr5 function in cre-expressing tissues. Examples listed below.
When bred to germline Cre-expressing mice (Stock No. 006054), the resulting Tlr5 global knockout homozygotes are expected to exhibit increased levels of flagellated bacteria in the intestine, that will promote low-grade inflammation, metabolic syndrome and colitis.
When Tlr5fl mice are bred to Villin-Cre transgenic mice (Stock No. 004586), the resulting Tlr5ΔIEC homozygotes lack TLR5 expression in intestinal intraepithelial cells, and exhibit a phenotype similar to global TLR5 knockout mice.
When Tlr5fl mice are bred to Cd11c-Cre transgenic mice (Stock No. 007567), the resulting Tlr5ΔDC homozygotes lack TLR5 expression in dendritic cells, and exhibit complete loss of flagellin-induced production of interleukin-22, but do not develop an inflammation-associated phenotype or an altered microbiota composition, as seen in Tlr5ΔIEC and in global TLR5 knockout mice.
A targeting vector was designed by Dr. Andrew T. Gewirtz and Benoit Chassaing (Georgia State University) to have a loxP site upstream of exon 4, and a frt::loxP::Neo::frt::loxP cassette was inserted 211 base pairs downstream of exon 4 of the toll-like receptor 5 gene (Tlr5) on chromosome 1.
The construct was electroporated into the C57BL/6N-derived Ingenious Targeting Laboratory, Inc. embryonic stem (ES) cell line iTL IC1. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric mice were bred with C57BL/6N to establish the colony.
Offspring were bred with C57BL/6-congenic Flp-expressing mice for germline deletion of the neo cassette; leaving exon 4 flanked by loxP sites.
The resulting TLR5fl mice were bred together for several generations (and the Flp-expressing transgene was removed). At least one generation of breeding to C57BL/6J also occurred in 2012. In 2016, black homozygous males were sent to The Jackson Laboratory Repository (see SNP results below).
Upon arrival, sperm was cryopreserved. To establish our live colony in 2016, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304).
In 2016, 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 males sent to The Jackson Laboratory Repository and a cohort of first generation rederived living mice. 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 in some mice. These data suggest that the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J;C57BL/6N genetic background (mostly C57BL/6J).
|Allele Name||targeted mutation 1.1, Andrew Gewirtz|
|Allele Type||Targeted (Conditional ready (e.g. floxed))|
|Gene Symbol and Name||Tlr5, toll-like receptor 5|
|Strain of Origin||C57BL/6NTac|
|Molecular Note||A targeting vector was designed to place a loxP site upstream of exon 4, and a frt::loxP::Neo::frt::loxP cassette inserted 211 base pairs downstream of exon 4. Flp-mediated recombination removed the FRT-flanked neo cassette leaving exon 4 floxed.|
When maintaining a live colony, homozygous mice may be bred together.
When using the TLR5fl (TLR5 floxed exon 4) mouse strain in a publication, please cite the originating article(s) and include JAX stock #028599 in your Materials and Methods section.