CD99L2 Mouse

What is mouse CD99L2 and how does it relate to CD99?

Mouse CD99L2 (CD99 antigen-like protein 2, also known as MIC2-like protein 1) is a Type I glycoprotein that belongs to the CD99 family. It is related to CD99 but shows only approximately 38% sequence identity . Both proteins are implicated in cellular adhesion and migration processes, with distribution patterns that largely overlap in various cell types . CD99L2 functions as a paralog of CD99, maintaining similar roles in leukocyte trafficking but with distinct molecular mechanisms.

What is the expression pattern of CD99L2 in mouse tissues?

CD99L2 is expressed on leukocytes and endothelial cells as well as several other cell types . Its distribution largely overlaps with CD99 with few exceptions, such as bone marrow-derived neutrophils that express CD99 but not CD99L2 . The protein is highly enriched at contact regions between cultured endothelial cells . Researchers can detect CD99L2 expression using immunoperoxidase histochemistry or immunofluorescence techniques with specific antibodies against mouse CD99L2 .

What are the recommended methods for detecting CD99L2 expression in mouse tissues?

For detecting CD99L2 expression in mouse tissues, researchers should consider:

• Immunohistochemistry: Using either immunoperoxidase histochemistry or immunofluorescence with validated antibodies against mouse CD99L2. Polyclonal antibodies against murine CD99L2 have been successfully employed .

• Flow cytometry: For analysis of CD99L2 expression on leukocyte subpopulations.

• Western blotting: To quantify protein expression levels in tissue lysates.

When conducting these experiments, it's critical to include appropriate controls, particularly CD99L2-deficient mouse tissues to confirm antibody specificity .

How can researchers generate recombinant mouse CD99L2 protein for functional studies?

Recombinant mouse CD99L2 protein can be expressed using human embryonic kidney (HEK) 293 cells with a histidine tag for purification purposes. The recombinant protein typically includes amino acids 1-164 of the mouse CD99L2 sequence . When producing recombinant CD99L2:

• Verify protein purity (aim for >98% purity)

• Ensure low endotoxin levels (<1 EU/μg)

• Validate functionality through appropriate binding or functional assays

• Confirm protein identity through mass spectrometry or sequencing

The recombinant protein can be used for antibody generation, binding studies, or in vitro functional assays .

What strategies have been used to generate CD99L2-deficient mice?

CD99L2-deficient mice have been generated using homologous recombination technologies. The targeting construct typically contains:

• Two homology arms flanking the target region

• A positive selection marker (e.g., PGK-Neo inserted in anti-sense orientation to minimize promoter interference)

• Deletion of the entire first coding exon of CD99L2 (124 nt) including the translation start codon and a portion of intron 1 (990 nt)

After electroporation into ES cells, G418-resistant clones are screened by PCR and Southern blotting for specific homologous recombination. Chimeric mice are then generated through blastocyst injection and breeding for germline transmission .

What is the baseline phenotype of CD99L2 knockout mice under normal conditions?

CD99L2-deficient mice display the following characteristics under normal conditions:

• Normal development and weight gain

• Normal lifespan in conventional barrier housing

• No tendency toward illness or wound healing difficulties

• No gross abnormalities of internal organs upon necropsy

• Normal complete blood counts including:

  • No differences in absolute numbers or percentage of white blood cell subtypes

  • Normal red blood cell parameters and platelet counts

Additionally, CD99L2 knockout mice maintain normal numbers of resident macrophages, lymphocytes, and mast cells in the peritoneal cavity under homeostatic conditions .

How does the absence of CD99L2 affect the expression of related adhesion molecules?

The constitutive absence of CD99L2 does not alter the expression of related adhesion molecules:

• CD99 levels on both endothelial cells and leukocytes remain unchanged

• PECAM-1 (CD31) expression is maintained at normal levels

• ICAM-1 expression patterns on endothelium remain similar to wild-type mice

• CD18 levels on leukocytes are unaffected

These findings indicate that CD99 does not increase to compensate for the absence of CD99L2, and there is no evident compensatory upregulation of other adhesion molecules .

What is the specific role of CD99L2 in leukocyte extravasation during inflammation?

CD99L2 plays a critical role in leukocyte extravasation, particularly at a late step in the process. Specifically:

• It helps leukocytes overcome the endothelial basement membrane

• Acts at the same site as PECAM-1 (CD31) but functions independently of it

• Serves as a homophilic adhesion molecule, though these interactions may not be required for cell aggregation

In CD99L2-deficient mice, the inflammatory response in the thioglycollate peritonitis model shows:

• Greater than 80% reduction in neutrophil infiltration

• Nearly complete blockage of monocyte emigration into the peritoneal cavity at 16 hours post-inflammatory challenge

How do neutrophils and monocytes differ in their dependence on CD99L2 for transendothelial migration?

Based on thioglycollate-induced peritonitis experiments in CD99L2-deficient mice:

• Neutrophil infiltration is blocked by approximately 83% compared to wild-type mice

• Monocyte influx is reduced almost to baseline levels (nearly complete inhibition)

This suggests that monocytes may be more dependent on CD99L2 for successful transendothelial migration than neutrophils . This differential dependence on CD99L2 could reflect distinct molecular requirements for different leukocyte subsets during the extravasation process.

What inflammatory models are appropriate for studying CD99L2 function in vivo?

Several inflammatory models have been used to study CD99L2 function:

• Thioglycollate peritonitis model: A well-established model of sterile inflammation induced by intraperitoneal injection of 4% thioglycollate broth. This allows assessment of both neutrophil (early) and monocyte (later) recruitment .

• Anti-CD99L2 antibody blocking studies: Administration of anti-CD99L2 antibodies has been shown to block the influx of neutrophils to inflammation sites .

• Intravital microscopy: Can be used to visualize leukocyte-endothelial interactions in real-time in CD99L2-deficient mice.

When using these models, researchers should consider timing (e.g., 16 hours post-challenge captures both neutrophil and monocyte responses), strain differences (e.g., FVB/n vs. C57BL/6), and comprehensive readouts including both cellular composition and inflammatory mediators .

How does CD99L2 interact with CD99, and what are the functional consequences?

CD99L2 can physically interact with CD99 to form heterodimers, in addition to forming homodimers. Key aspects of this interaction include:

• The interaction is cytoplasmic domain-dependent

• Heterodimers between CD99 and CD99L2 localize more efficiently to the plasma membrane than homodimers

• The interaction enhances CD99L2 trafficking to the plasma membrane, regardless of whether CD99L2 is transiently overexpressed or endogenously expressed

This interaction can be analyzed using techniques such as bimolecular fluorescence complementation, immunoprecipitation, and fluorescence resonance energy transfer assays .

What experimental approaches can be used to study CD99L2 dimerization and interactions?

Several experimental approaches are suitable for investigating CD99L2 dimerization and molecular interactions:

• Bimolecular fluorescence complementation (BiFC): To visualize protein-protein interactions in living cells

• Co-immunoprecipitation: To confirm physical association between CD99L2 and potential binding partners

• Fluorescence resonance energy transfer (FRET): To study proximity and interaction dynamics

• Cell aggregation assays: To assess functional consequences of CD99L2 expression and dimerization

• Membrane trafficking studies: To evaluate the effect of interactions on subcellular localization

These approaches can help elucidate the molecular mechanisms by which CD99L2 contributes to leukocyte adhesion and transmigration.

How does mouse CD99L2 compare to its human counterpart in structure and function?

Mouse CD99L2 and human CD99L2 share moderate sequence homology . While both proteins are implicated in leukocyte adhesion and migration, there may be species-specific differences in:

• Tissue distribution patterns

• Regulatory mechanisms

• Interaction partners

• Roles in specific inflammatory conditions

Understanding these similarities and differences is crucial for translating findings from mouse models to human disease contexts. Additional comparative studies using recombinant proteins from both species and cross-species cellular assays would help clarify the translational relevance.

Can findings from CD99L2 mouse models be applied to human inflammatory diseases?

While mouse CD99L2 models provide valuable insights into basic mechanisms of leukocyte extravasation, several considerations are important when translating these findings to human inflammatory diseases:

• The moderate sequence homology between mouse and human CD99L2 suggests potentially conserved functions

• CD99L2's role in leukocyte extravasation across the endothelial basement membrane represents a fundamental biological process likely conserved across species

• The non-redundant role of CD99L2 in inflammatory responses in mice suggests it may be a viable therapeutic target in humans

What are common challenges in generating and validating antibodies against mouse CD99L2?

Researchers may encounter several challenges when generating and validating antibodies against mouse CD99L2:

• Cross-reactivity with CD99 due to structural similarities despite limited sequence homology

• Potential glycosylation differences between recombinant and native CD99L2

• Conformational epitopes that may be lost in fixed tissues

• Strain-specific variations in CD99L2 expression

To address these challenges:

• Validate antibodies using CD99L2-deficient tissues as negative controls

• Compare multiple antibodies targeting different epitopes

• Use both polyclonal and monoclonal antibodies when available

• Verify specificity in multiple applications (IHC, flow cytometry, Western blot)

How should researchers account for strain differences when studying CD99L2 function?

CD99L2 function has been studied in different mouse strains, including C57BL/6 and FVB/n, with potentially different outcomes. Key considerations include:

• Backcrossing CD99L2-deficient mice into the desired strain for at least 9 generations to achieve genetic homogeneity

• Using age- and sex-matched littermate controls whenever possible

• Conducting comparative studies across different strains to identify strain-dependent effects

• Considering the inflammatory model used, as strain differences may be more pronounced in certain contexts

For example, one study reported a 20-40% reduction in neutrophil or T cell emigration in C57BL/6 CD99L2-deficient mice, while another found >80% inhibition of neutrophil recruitment in FVB/n CD99L2-deficient mice .