The MyD88LSL knock-in/knock-out allele is a functional null prior to Cre recombinase exposure. These mice allow Cre recombinase-inducible restoration of functional MYD88 expression. MyD88LSL mice may be useful for studying cell-type-specific function of MYD88 in immune system abnormalities, as well as hematopoietic system and apoptotic abnormalities.
Bernhard Holzmann, Technische Universität München (Technical University Munich)
The MyD88LSL knock-in/knock-out allele has a floxed transcriptional termination element (loxP::En2 splice-acceptor::3xSTOP::IRES::lacZ-pA::DSE::loxP-NeoR-loxP) inserted between exons 1 and 2 that prevents transcription/translation of MYD88. Following cre-mediated recombination that removes the entire floxed region, MYD88 expression and function are fully recovered.
Homozygous (MyD88LSL/LSL) cells have no detectable MYD88 protein expression, and are expected to exhibit the MYD88 knockout phenotype (described below). When maintained at high health status (specific pathogen-free), the donating investigator reports that MyD88LSL/LSL mice are viable and fertile with normal lifespan. While the MyD88LSL allele contains an IRES::lacZ-pA sequence, the expression of β-galactosidase has not been characterized to date (April 2016).
Following Cre recombinase exposure, one of three resulting outcomes may be possible: [i] the entire floxed region is deleted, [ii] only the En2 splice-acceptor::3xSTOP::IRES::lacZ-pA::DSE region is deleted or [iii] only the neo selection cassette is deleted. Genotype outcome i, called MyD88Lox or MyD88ON, results in restoration of MYD88 expression/function. To date (April 2016), it has not been determined if genotype outcomes ii or iii are produced.
Specifically, when MyD88LSL/LSL mice are bred to LysMcre knockin/knockout mice (Stock No. 004781), the resulting MyD88MYEL mice exhibit restored expression and function of MYD88 in macrophages/granulocytes.
MYD88 is a cytosolic adapter protein that plays a central role in the innate and adaptive immune response. MYD88 signaling is required to limit bacterial burden and prolong survival during infection. Global MYD88-deficiency is associated with increased susceptibility to bacterial and viral infections, a number of immune system abnormalities, as well as hematopoietic system, molecular signaling and apoptotic abnormalities. As such, and similar to other immunodeficient strains, maintaining MYD88-deficient mice in high health status (specific pathogen-free) vivaria promotes overall colony health. If MYD88-deficient animals are maintained in low health barrier rooms, the use of medicated water (e.g., sulfatrim/trimethoprim-sulfa or enrofloxacin/Baytril) may be warranted to increase overall colony health. In general, global MYD88-deficiency protects mice from LPS-induced septic shock and attenuates systemic hyperinflammation and lethality in murine sepsis models.
The MyD88LSL allele was created in the laboratory of Dr. Bernhard Holzmann (Technische Universität München) by inserting a floxed transcriptional termination element between exons 1 and 2 of the myeloid differentiation primary response gene 88 locus (Myd88) on chromosome 9. The targeting vector contained (from 5' to 3') a loxP site, an engrailed-2 splice acceptor site followed by stop codons in all reading frames (En-2/stp), an internal ribosomal entry site (IRES), a β-galactosidase sequence followed by a polyadenylation signal (lacZ-pA), a downstream element (DSE) and a loxP-flanked neomycin resistance cassette. The construct was electroporated into 129P2/OlaHsd-derived E14.1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric mice were bred with C57BL/6N mice for germline transmission and to establish the MyD88LSL mice. The donating investigator reported that the MyD88LSL colony was backcrossed to C57BL/6N for a total of more than ten generations prior to sending heterozygous males to The Jackson Laboratory Repository in 2016 (see SNP results below).
Upon arrival, males were bred to C57BL/6NJ females (Stock No. 005304) for one generation. By June 2017, the colony was additionally backcrossed to C57BL/6NJ at least two generations. After this, the MyD88LSL mice were made available for distribution.
Of note, at least once in the colony pedigree, a MyD88LSL female was bred to a C57BL/6NJ inbred male (thus the Y chromosome of the congenic strain is of C57BL/6NJ origin).
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 mice sent to The Jackson Laboratory Repository and our first generation rederived living colony. While 26 of 27 markers throughout the genome suggested a C57BL/6 genetic background, one marker on chromosome 13 [~15cM] was segregating for C57BL/6;129 in some mice. In addition, 2-4 markers that determine C57BL/6J from C57BL/6N were found to be segregating in some mice (chromosomes 8 [~15.2 Mbp], 11 [~4.4 Mbp], 15 [~57.6 Mbp] and 19 [~49.9 Mbp]). Collectively, these data suggest that the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J;C57BL/6N genetic background. The C57BL/6;129 segregation on chromosome 13 is unexplained.
In August 2017, a 134 SNP panel assay was performed on a cohort of 41 mice. It showed >98% of the markers to be C57BL/6 allele-type. Using this data, breeders were chosen that were C57BL/6 allele-type at 100% of the markers (including all four markers on chromosome 13).
|Allele Name||targeted mutation 1, Bernhard Holzmann|
|Allele Type||Targeted (Conditional ready (e.g. floxed), Null/Knockout)|
|Allele Synonym(s)||Myd88LSL; MyD88OFF|
|Gene Symbol and Name||Myd88, myeloid differentiation primary response gene 88|
|Strain of Origin||129P2/OlaHsd|
|Molecular Note||An intron gene trap cassette, containing a loxP site, Engailed 2 splice acceptor/STOP (En-2/stp), IRES, lacZ, downstream element (DSE) and floxed neo cassette, was targeted to intron 1. This allele is both a knock-out and conditional-ready allele, as it can be reverted back to wild-type expression through cre-mediated recombination. Western blot analysis confirmed the absence of protein expression in bone marrow derived macrophage stimulated with (S)-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH (P3C).|
When maintaining a live colony, heterozygous mice may be bred together, to wildtype mice from the colony or to C57BL/6NJ inbred mice (Stock No. 005304). Alternatively, homozygous mice may be bred together (see below).
When maintained at high health status (specific pathogen-free), the donating investigator reports that homozygous (MyD88LSL/LSL) mice are viable and fertile with normal lifespan, and may be bred together. Importantly, global MYD88-deficiency is associated with increased susceptibility to bacterial and viral infections, a number of immune system abnormalities, as well as hematopoietic system, molecular signaling and apoptotic abnormalities. As such, and similar to other immunodeficient strains, maintenance in high health status (specific pathogen-free) vivaria promotes overall colony health. If MYD88-deficient animals are maintained in low health barrier rooms, the use of medicated water (e.g., sulfatrim/trimethoprim-sulfa or enrofloxacin/Baytril) may be warranted to increase overall colony health.
When using the MyD88LSL mouse strain in a publication, please cite the originating article(s) and include JAX stock #028939 in your Materials and Methods section.