MERTK Mouse

What is MERTK and what are the available MERTK mouse models?

MERTK is a receptor tyrosine kinase (RTK) that belongs to the TAM family, which also includes TYRO3 and AXL receptors. The name MERTK was derived from its expression pattern in monocytes, epithelial tissues, and reproductive tissues, as well as it being a tyrosine kinase . Currently, there are multiple MERTK knockout mouse models available to researchers. The original and most widely used model is referred to as Mertk-/-V1, which was generated by Camenisch et al. in 1999 using 129P2/OlaHsd-derived embryonic stem cells that were injected into C57BL/6 (B6) blastocysts and subsequently backcrossed to B6 mice . This line is available through The Jackson Laboratory (strain #011122). More recently, two additional MERTK knockout models have been developed: Mertk-/-V2, generated by breeding Mertk floxed mice with Rosa26 ERT2Cre+ mice to achieve germline inactivation, and Mertk-/-V3, generated using CRISPR/Cas9-mediated genome engineering in C57BL/6NJ eggs . These newer models differ significantly from the original in their genetic backgrounds and the resulting phenotypes they exhibit.

How do I confirm complete MERTK deletion in my experimental model?

Confirming complete MERTK deletion requires a multi-faceted approach to ensure the validity of your experiments. First, genomic DNA analysis should be performed using PCR with primers specific to the targeted region of the Mertk gene . For the Mertk-/-V1 model, this involves examining the neomycin resistance cassette inserted to disrupt the gene, while for Mertk-/-V2, verification of excision of exon 18 (encoding the kinase domain) should be confirmed . In the Mertk-/-V3 model, confirmation of the frameshift mutation in exons 3 and 4 is necessary . Beyond genomic verification, RNA expression analysis using qPCR or RNA sequencing should be conducted to confirm the absence of Mertk transcript . Additionally, protein-level verification is crucial and can be accomplished through Western blotting or immunohistochemistry using validated anti-MERTK antibodies in tissues known to express MERTK, such as retinal pigment epithelium, macrophages, or monocytes. Finally, functional assays measuring MERTK-dependent processes like phagocytosis capacity in relevant cell types provide further confirmation of complete functional deletion.

What control mice should I use for MERTK knockout experiments?

Selecting appropriate controls for MERTK knockout experiments is critical given the recent discoveries about genetic modifiers affecting Mertk-/-V1 phenotypes. For the original Mertk-/-V1 model, the ideal controls are wild-type littermates derived from heterozygous breeding pairs to ensure matching genetic backgrounds, particularly for the 129P2-derived region on chromosome 2 that contains modifier alleles . When using newer models like Mertk-/-V2 or Mertk-/-V3, wild-type C57BL/6J or C57BL/6NJ mice (matching the background of your specific model) should be used as controls . In experiments comparing different MERTK knockout models, it becomes essential to include wild-type controls for each model to account for strain-specific differences. For conditional knockout models, proper controls should include Cre-negative floxed mice (Mertk f/f) and possibly Cre-positive wild-type mice to control for potential Cre toxicity effects . Additionally, in cases where the effects of both MERTK and TYRO3 are being investigated, appropriate single knockout controls should be included alongside double knockouts to delineate the contribution of each receptor.

What is the role of genetic modifiers in MERTK knockout phenotypes?

Genetic modifiers play a crucial role in determining MERTK knockout phenotypes, as recently demonstrated through comprehensive genetic analysis. The original Mertk-/-V1 mouse carries a significant 15.08 cM region between D2Mit206 and D2Mit168 on chromosome 2 that is of 129P2 origin, even after extensive backcrossing to C57BL/6 . This region contains numerous genes, including the MERTK paralog Tyro3, that are differentially expressed compared to pure C57BL/6 backgrounds . Through detailed genetic studies, researchers discovered that the retinal degeneration phenotype in Mertk-/-V1 mice requires the simultaneous loss of function of both Mertk and Tyro3, with Tyro3 providing functional redundancy for photoreceptor outer segment phagocytosis in retinal pigment epithelium . Short tandem repeat (STR) analysis confirmed that this 129P2-derived genomic segment is absent in the newer Mertk-/-V2 and Mertk-/-V3 models, which have entirely C57BL/6-derived chromosome 2 . These findings have profound implications for interpreting previous research using Mertk-/-V1 mice and underscore the importance of considering genetic background effects when attributing phenotypes to specific gene deletions.

How should I design experiments to validate previous findings using MERTK knockout mice?

Designing validation experiments for previous MERTK knockout findings requires careful consideration of genetic background effects and model selection. First, researchers should clearly identify which MERTK knockout model was used in the original studies (most commonly Mertk-/-V1) and obtain the appropriate models for comparison . When validating findings from Mertk-/-V1 studies, it is advisable to include both the original Mertk-/-V1 model and one of the newer models (Mertk-/-V2 or Mertk-/-V3) to determine whether phenotypes are reproducible across different genetic backgrounds . Comprehensive genotyping should be performed to confirm the genetic background of each model, particularly focusing on known modifier regions such as the 129P2-derived segment on chromosome 2 in Mertk-/-V1 mice . RNA sequencing or targeted gene expression analysis should be conducted to identify differentially expressed genes between models that might contribute to phenotypic differences. For studies involving retinal degeneration, the status of Tyro3 should be specifically assessed, considering its now-established role as a genetic modifier . Finally, when validating anti-tumor immunity findings, multiple tumor models should be tested, and detailed immune profiling should be performed to characterize the mechanisms underlying any observed differences in tumor resistance.

What techniques are most reliable for analyzing MERTK-dependent phagocytosis?

Analyzing MERTK-dependent phagocytosis requires sophisticated techniques that capture the nuances of this cellular process. For in vitro studies, flow cytometry-based phagocytosis assays using fluorescently labeled targets (such as pHrodo-labeled apoptotic cells or photoreceptor outer segments) provide quantitative measurements of phagocytic capacity in MERTK-expressing cells compared to MERTK-deficient counterparts . Confocal microscopy with Z-stack imaging offers complementary visual confirmation of particle internalization, distinguishing between surface-bound and truly engulfed targets. For in vivo analysis of retinal phagocytosis, immunohistochemical staining of retinal pigment epithelium (RPE) for markers of phagocytosis (such as rhodopsin-positive phagosomes) at specific times during the diurnal cycle provides insights into the efficiency of photoreceptor outer segment clearance . Electron microscopy remains the gold standard for detailed ultrastructural analysis of phagosomes in RPE cells. When comparing phagocytosis between different MERTK knockout models, it is essential to consider the functional redundancy with TYRO3, particularly in models where TYRO3 expression is preserved . Finally, complementation experiments reintroducing wild-type or mutant MERTK constructs into MERTK-deficient cells can definitively establish the specific role of MERTK in observed phagocytosis defects.

How can I distinguish between MERTK's phagocytic and anti-inflammatory functions?

Distinguishing between MERTK's phagocytic and anti-inflammatory functions requires specialized experimental approaches that can separate these interconnected processes. To assess MERTK's anti-inflammatory function, researchers should measure inflammatory cytokine production (such as TNFα, IL-6, IL-1β) following controlled immune challenges with reagents like LPS or poly(I:C) in MERTK-sufficient versus MERTK-deficient cells or animals . Phosphorylation analysis of downstream signaling components, particularly STAT1 and SOCS proteins, provides mechanistic insights into MERTK-mediated suppression of inflammatory responses. For phagocytic function assessment, specialized assays using fluorescently labeled apoptotic cells, photoreceptor outer segments, or other physiologically relevant targets should be employed . To distinguish between these functions, structure-function studies using MERTK mutants with selective defects in phagocytosis or anti-inflammatory signaling can be particularly informative. Additionally, conditional knockout models targeting specific MERTK-expressing cell populations can help delineate cell type-specific contributions to observed phenotypes. When interpreting results, researchers should consider the potential functional redundancy with other TAM receptors (TYRO3 and AXL), which share overlapping ligands and signaling pathways but may contribute differentially to phagocytosis versus anti-inflammatory functions in different tissue contexts .

How does the genetic background affect interpretation of MERTK knockout phenotypes?

The genetic background profoundly impacts the interpretation of MERTK knockout phenotypes, as demonstrated by recent comparative studies. The original Mertk-/-V1 model, despite being backcrossed >10 generations into C57BL/6 mice, retains a 15.08 cM region on chromosome 2 that is derived from the 129P2 background of the original embryonic stem cells . This region contains numerous genes that show differential expression compared to pure C57BL/6 backgrounds, including the MERTK paralog Tyro3 . RNA sequencing analysis revealed significant transcriptional differences between Mertk-/-V1, Mertk-/-V2, and Mertk-/-V3 mice, with many differentially expressed genes in Mertk-/-V1 mice that were not altered in the newer models . These genetic differences explain why phenotypes such as retinal degeneration and anti-tumor immunity observed in Mertk-/-V1 mice are not reproduced in the newer models despite confirmed MERTK deletion . The implications are substantial: many traits previously attributed solely to MERTK deficiency are now understood to be products of epistatic interactions with modifiers in the 129P2 genome . Researchers must therefore exercise caution when interpreting historical data from Mertk-/-V1 mice and should validate key findings using multiple MERTK knockout models to distinguish MERTK-specific effects from background-dependent phenotypes.

What tools are available for assessing genetic modifiers in MERTK mouse models?

Multiple sophisticated tools are available for assessing genetic modifiers in MERTK mouse models, enabling researchers to distinguish true MERTK-dependent phenotypes from background effects. Short tandem repeat (STR) analysis can be employed to identify genomic regions of non-C57BL/6 origin in backcrossed knockout mice, as demonstrated in the analysis of Mertk-/-V1 mice where a 15.08 cM region between D2Mit206 and D2Mit168 on chromosome 2 was identified as being of 129P2 origin . Genome-wide single-nucleotide polymorphism (SNP) analysis provides a comprehensive assessment of genetic background composition, as employed for characterizing the Mertk-/-V2 and Mertk-/-V3 models where the percentage of C57BL/6J versus C57BL/6NJ genome was determined . RNA sequencing represents a powerful approach for identifying differentially expressed genes in various MERTK knockout models compared to wild-type controls, potentially revealing modifier genes that contribute to phenotypic differences . Quantitative trait locus (QTL) mapping using specifically designed crosses between different strains can help localize regions harboring modifier genes affecting phenotypes of interest. Finally, CRISPR/Cas9-mediated genome editing can be used to specifically modify candidate modifier genes identified through these approaches, allowing direct testing of their contributions to observed phenotypes.

How can I distinguish between phenotypes caused by MERTK deficiency versus background effects?

Distinguishing between phenotypes caused by MERTK deficiency versus background effects requires a multi-pronged experimental approach. First, researchers should compare phenotypes across multiple independently generated MERTK knockout models with different genetic backgrounds, such as the original Mertk-/-V1 model versus the newer Mertk-/-V2 and Mertk-/-V3 models . Phenotypes that are consistently observed across all models despite their different genetic backgrounds can be more confidently attributed to MERTK deficiency alone. For phenotypes observed only in specific models (like the retinal degeneration in Mertk-/-V1), genetic complementation tests should be performed to identify potential modifier genes . This can involve creating compound mutants with targeted deletions of candidate modifier genes (such as Tyro3) on a pure C57BL/6 background . Genomic rescue experiments, where wild-type MERTK is reintroduced into MERTK-deficient cells or animals through transgenic approaches, can confirm whether phenotypes are directly caused by MERTK deficiency. Single-cell transcriptomic analysis of relevant tissues from different MERTK knockout models can reveal cell type-specific effects and identify pathways differentially affected by background genetics. Finally, mechanistic studies tracing the molecular consequences of MERTK deletion in different models can help establish causal relationships between MERTK deficiency and observed phenotypes.

What novel approaches might better elucidate MERTK functions in specific tissues?

Novel approaches to elucidate tissue-specific MERTK functions should leverage cutting-edge technologies while addressing the complexities revealed by recent research. Inducible, tissue-specific CRISPR-Cas9 systems allow temporal and spatial control of MERTK deletion, avoiding developmental compensation that might mask phenotypes in germline knockouts . Single-cell multi-omics approaches combining transcriptomics, proteomics, and epigenomics can reveal cell type-specific MERTK functions and identify compensatory mechanisms in different genetic backgrounds. Organoid models derived from various MERTK knockout mice or human cells with MERTK mutations provide three-dimensional tissue contexts for studying MERTK functions in development and disease. In vivo imaging techniques using fluorescently tagged MERTK ligands or downstream signaling reporters enable real-time visualization of MERTK activity in intact tissues. For retinal research specifically, adaptive optics scanning light ophthalmoscopy offers unprecedented resolution for monitoring progressive changes in retinal structure in different MERTK mouse models . Spatially resolved transcriptomics can map gene expression changes in specific retinal layers following MERTK deletion. Finally, cross-species comparative studies examining MERTK functions in diverse organisms can identify evolutionarily conserved versus species-specific roles, potentially explaining discrepancies between mouse models and human disease presentations.