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.
"MICE" means mouse strains, their progeny derived by inbreeding or crossbreeding, unmodified derivatives from mouse strains or their progeny supplied by The Jackson Laboratory ("JACKSON"). "PRODUCT(S)" means biological materials supplied by JACKSON, and their derivatives. "SERVICES" means projects conducted by JACKSON for other parties that may include but are not limited to the use of MICE or PRODUCTS. "RECIPIENT" means each recipient of MICE, PRODUCTS, or SERVICES provided by JACKSON including each institution, its employees and other researchers under its control. MICE or PRODUCTS shall not be: (i) used for any purpose other than internal research, (ii) sold or otherwise provided to any third party for any use, or (iii) provided to any agent or other third party to provide breeding or other services. Acceptance of MICE, PRODUCTS or SERVICES from JACKSON shall be deemed as agreement by RECIPIENT to these conditions, and departure from these conditions requires JACKSON’s prior written authorization.
MICE, PRODUCTS AND SERVICES ARE PROVIDED "AS IS". JACKSON EXTENDS NO WARRANTIES OF ANY KIND, EITHER EXPRESS, IMPLIED, OR STATUTORY, WITH RESPECT TO MICE, PRODUCTS OR SERVICES, INCLUDING ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, OR ANY WARRANTY OF NON-INFRINGEMENT OF ANY PATENT, TRADEMARK, OR OTHER INTELLECTUAL PROPERTY RIGHTS.
In case of dissatisfaction for a valid reason and claimed in writing by a purchaser within ninety (90) days of receipt of, PRODUCTS or SERVICES, JACKSON will, at its option, provide credit or replacement for the PRODUCT received or the SERVICES provided; JACKSON makes no other representations and this shall be the exclusive remedy of the purchaser. Please note specific policy for live mice.
Consistent with the requirement for a written understanding regarding animal care and use, the JACKSON Animal Care and Use Committee will review the animal care and use protocol(s) associated with any SERVICES to be performed at JACKSON, and JACKSON shall have ultimate responsibility and authority for the care of animals while on site or in JACKSON custody.
In no event shall JACKSON, its trustees, directors, officers, employees, and affiliates be liable for any causes of action or damages, including any direct, indirect, special, or consequential damages, arising out of the provision of MICE, PRODUCTS, or SERVICES, including economic damage or injury to property and lost profits, and including any damage arising from acts or negligence on the part of JACKSON, its agents or employees. Unless prohibited by law, in purchasing or receiving MICE, PRODUCTS, or SERVICES from JACKSON, purchaser or recipient, or any party claiming by or through them, expressly releases and discharges JACKSON from all such causes of action or damages, and further agrees to defend and indemnify JACKSON from any costs or damages arising out of any third party claims.
MICE, PRODUCTS or SERVICES are to be used in a safe manner and in accordance with all applicable governmental rules and regulations.
The foregoing represents the General Terms and Conditions applicable to JACKSON’s MICE, PRODUCTS or SERVICES. In addition, special terms and conditions of sale of certain MICE, PRODUCTS, or SERVICES may be set forth separately in JACKSON web pages, catalogs, price lists, contracts, and/or other documents, and these special terms and conditions shall also govern the sale of these MICE, PRODUCTS and SERVICES by JACKSON, and by its licensees and distributors.
Acceptance of delivery of MICE, PRODUCTS or SERVICES shall be deemed agreement to these terms and conditions. No purchase order or other document transmitted by purchaser or recipient that may modify the terms and conditions hereof, shall be in any way binding on JACKSON, and instead the terms and conditions set forth herein, including any special terms and conditions set forth separately, shall govern the sale of MICE, PRODUCTS or SERVICES by JACKSON.