Lyz2-IRES-CreERT2 Mice

Catalog Number: C001499

Strain Name: C57BL/6JCya-Lyz2^{em3(IRES-CreERT2)}/Cya

Genetic Background: C57BL/6JCya

Reproduction: Homozygote x Homozygote

Strain Description

Lysozyme, encoded by the LYZ gene, is an integral component of the innate immune system. Its primary function is to disrupt bacterial cell walls by cleaving the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in bacterial peptidoglycan, leading to bacterial lysis. Lysozyme is abundant in various secretions, including tears, saliva, human milk, and mucus, as well as in the cytoplasmic granules of macrophages and polymorphonuclear neutrophils. Cytoplasmic lysozyme expression is high in bone marrow cells, Paneth cells, and a subset of salivary gland cells ^[1,2].

Mice have two lysozyme-encoding genes, Lyz1 and Lyz2, which are highly homologous to the human LYZ gene. Lyz1 is primarily expressed in Paneth cells, while Lyz2 is mainly expressed in myeloid cells. Myeloid-specific expression of Lyz2 is associated with cell-type-specific demethylation of its downstream enhancer. The Lyz2 protein also has lysozyme activity and participates in defense responses against both Gram-negative and Gram-positive bacteria. Lyz2 is expressed in the hematopoietic system, liver, mesothelium, small intestine, and yolk sac of mice ^[3,4].

Lyz2-IRES-CreERT2 mice were generated using gene editing technology to integrate the tamoxifen-inducible CreERT2 recombinase expression element into the 3’UTR of the mouse Lyz2 gene. This mimics the expression pattern of the endogenous gene while maintaining Lyz2 expression. When bred with mice carrying a loxP site-flanked sequence, Cre recombinase-mediated recombination of the flanked sequence occurs in myeloid cells, including monocytes, mature macrophages, and granulocytes, following tamoxifen induction.

Strain Strategy

The IRES-CreERT2 gene expression element was integrated into the 3'UTR region of the mouse Lyz2 gene using gene editing technology.

Validation Data

a. Method

Lyz2-IRES-CreERT2 mice were mated with Rosa26-LSL-tdTomato mice to generate double heterozygous zygote mice. Tamoxifen induction (3 mg/mouse for 3 days, i.p.) was performed at 8 weeks of age, resulting in Cre recombinase-mediated deletion of the LSL elements and subsequent expression of tdTomato in Cre-positive cells. One week after induction, peripheral blood was collected from the offspring, and the distribution of tdTomato was determined by flow cytometry to assess Cre recombinase expression. The control group received the same dose of corn oil.

b. Groups

Cre+Tam+: Lyz2-IRES-CreERT2[KI/+];Rosa26-LSL-tdTomato[CKI/+], tamoxifen-induced;

Cre+Tam-: Lyz2-IRES-CreERT2[KI/+];Rosa26-LSL-tdTomato[CKI/+], corn oil-treated;

Cre-Tam+: Rosa26-LSL-tdTomato[CKI/CKI], tamoxifen-induced.

c. Result

1) Cre recombinase expression in peripheral blood

Figure 2. FACS analysis of peripheral blood macrophages. Cre recombinase activity and specificity were assessed in mouse peripheral blood macrophages (CD45+Cd11b+) by FACS analysis of tdTomato protein expression. Strong Cre recombinase activity was detected in macrophages from tamoxifen-induced double heterozygous mice (Cre+Tam+), with more than 90% of macrophages tdTomato-positive. However, Cre recombinase activity was undetectable in both tamoxifen-untreated double heterozygous mice (Cre+Tam-) and tamoxifen-treated Rosa26-LSL-tdTomato mice (Cre-Tam+). These results demonstrate high Cre recombinase activity and specificity under this induction regimen, with no evidence of expression leakage.

d. Summary

In Lyz2-IRES-CreERT2 mice, Cre recombinase activity is extremely high in peripheral blood macrophages, with no evidence of pre-induction leaky expression. Our internal data show that Cre recombinase recombination efficiency in macrophages can exceed 90% following the experimental induction protocol. However, recombination efficiency drops to approximately 13% 14 days after induction is complete. Additionally, when the induction protocol is adjusted to 2 mg tamoxifen per mouse per day for 3 or 5 days, or 4 mg tamoxifen per mouse per day for 3 days, recombination efficiency in peripheral blood CD45+Cd11b+ cells is approximately 40% one week after induction is complete (data not shown).

Announcements

The Cre recombinase expression element integration site of this strain is located on chromosome 10, please avoid using flox mice with the transgenic locus on chromosome 10 for breeding.

References

[1]Yoshimura K, Toibana A, Nakahama K. Human lysozyme: sequencing of a cDNA, and expression and secretion by Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1988 Jan 29;150(2):794-801. 
[2]Sharon N. The chemical structure of lysozyme substrates and their cleavage by the enzyme. Proc R Soc Lond B Biol Sci. 1967 Apr 18;167(1009):402-15.
[3]Peters CW, Kruse U, Pollwein R, Grzeschik KH, Sippel AE. The human lysozyme gene. Sequence organization and chromosomal localization. Eur J Biochem. 1989 Jul 1;182(3):507-16. 
[4]Clausen BE, Burkhardt C, Reith W, Renkawitz R, Förster I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res. 1999 Aug;8(4):265-77.