CREG1 Mouse
Description
Introduction to CREG1 Mouse Models
CREG1 (Cellular Repressor of E1A-Stimulated Genes 1) mouse models are genetically engineered systems used to study the glycoprotein’s roles in lysosomal biogenesis, autophagy, metabolism, and organ-specific pathophysiology. These models include global knockouts (KO), tissue-specific deletions (e.g., hepatocyte, cardiomyocyte, skeletal muscle), and transgenic overexpression lines. CREG1 is ubiquitously expressed in tissues such as the liver, spleen, heart, and adipose tissue, with critical roles in embryonic development and stress response .
Key Genetic Strategies
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Global KO: Creg1⁻/⁻ mice exhibit embryonic lethality by E7.5–E9.5 due to impaired endosomal-lysosomal function .
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Tissue-Specific KO:
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Transgenic Overexpression: Improved autophagy and cardiac function in diabetic cardiomyopathy models .
Table 1: CREG1 Antibody Validation in Mouse Tissues (Proteintech, 12220-1-AP)
| Application | Dilution | Detected Tissues/Cells |
|---|---|---|
| Western Blot (WB) | 1:500–1:1,000 | Jurkat cells, K-562 cells, mouse spleen |
| Immunofluorescence | 1:50 | Endosomal-lysosomal compartments |
Cardiac Function
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KO Phenotype:
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Overexpression: Rescued diabetic cardiomyopathy by enhancing autophagy (Creg1-TG mice) .
Hepatic Metabolism
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Hepatocyte KO (Creg1Δhep):
Skeletal Muscle Homeostasis
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Muscle-Specific KO:
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Therapeutic Intervention: Recombinant CREG1 restored mitochondrial density (+40%) and exercise capacity .
Metabolic and Obesity-Related Phenotypes
Mechanistic Insights
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Lysosomal Function: CREG1 promotes lysosomal acidification and autophagic degradation via interactions with mannose-6-phosphate receptors (M6PR) .
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Transcriptional Regulation: Binds to exocyst complex Sec8 to modulate endocytic trafficking .
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Developmental Role: Global KO causes embryonic lethality with vacuolar defects in visceral endoderm cells .
Therapeutic Potential
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Recombinant CREG1: Improved skeletal muscle mitochondrial function and exercise tolerance in aged Creg1;Ckm-Cre mice .
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Adipose Tissue Targeting: Overexpression in white adipose tissue induced beiging and UCP1-dependent thermogenesis, reducing diet-induced obesity .
Unresolved Questions and Future Directions
Product Specs
Recombinant CREG1 Mouse protein, expressed in E. coli, is a single polypeptide chain with a molecular weight of 24kDa. It encompasses 213 amino acids, spanning from position 32 to 220. This protein is engineered with a 24 amino acid His-tag fused to its N-terminus and is purified using proprietary chromatographic techniques.
The CREG1 solution is provided at a concentration of 1mg/ml and is formulated in a buffer consisting of 20mM Tris-HCl (pH 8.0), 10% glycerol, and 0.15M NaCl.
For short-term storage (up to 2-4 weeks), the product can be stored at 4°C. For extended storage, it is recommended to freeze the product at -20°C. The addition of a carrier protein (0.1% HSA or BSA) is advisable for long-term storage.
Repeated freezing and thawing of the product should be avoided.
Protein CREG1, Cellular repressor of E1A-stimulated genes 1, Creg1, Creg.
MGSSHHHHHH SSGLVPRGSH MGSMRGGRDH GDWDVDRRLP PLPPREDGPR VARFVTHVSD WGSLATISTI KEVRGWPFAD IISISDGPPG EGTGEPYMYL SPLQQAVSDL QENPEATLTM SLAQTVYCRN HGFDPQSPLC VHIMMSGTVT KVNKTEEDYA RDSLFVRHPE MKHWPSSHNW FFAKLKISRI WVLDYFGGPK VVTPEEYFNV TLQ.
Q&A
What genetic mouse models are available for studying CREG1 function?
Several genetically modified mouse models have been developed to study CREG1 function in specific tissues:
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CREG1-floxed mice (Creg1^fl/fl): These mice contain loxP sites flanking critical CREG1 exons, enabling tissue-specific deletion when crossed with appropriate Cre-expressing lines .
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Skeletal muscle-specific knockout mice (creg1;Ckm-Cre): Generated by crossing Creg1^fl/fl with transgenic mice expressing Cre recombinase under the muscle-specific creatine kinase (Ckm) promoter .
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Cardiac-specific knockout mice (Creg1-CKO): Created by crossing Creg1^fl/fl with mice expressing Cre under the cardiac-specific α-MHC promoter .
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CREG1 transgenic mice (Creg1-TG): Overexpress CREG1 systemically .
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Adipocyte P2-CREG1-transgenic mice (aP2-CREG1-Tg): Overexpress CREG1 specifically in adipose tissues .
These models enable investigation of CREG1's tissue-specific functions while circumventing the embryonic lethality observed in global CREG1 knockout mice.
How can I verify successful genetic modification in CREG1 mouse models?
Verification of CREG1 genetic modification requires multiple approaches:
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Genotyping: PCR amplification of genomic DNA from tail biopsies using primers specific for the modified locus. This confirms the presence of floxed alleles or Cre recombinase .
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Protein expression verification: Western blotting of tissue lysates using CREG1-specific antibodies. For tissue-specific models, compare target tissue with non-target tissues as internal controls .
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mRNA expression analysis: Quantitative RT-PCR of target tissues to confirm altered Creg1 transcript levels. In knockout models, expression should be significantly reduced, while in transgenic models, expression should be elevated compared to wild-type controls .
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Functional validation: Assess whether the expected physiological changes are observed. For example, skeletal muscle-specific knockout mice should show reduced exercise capacity .
How does CREG1 deficiency affect skeletal muscle function?
CREG1 deficiency has significant detrimental effects on skeletal muscle function:
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Muscle-specific creg1 knockout mice (creg1;Ckm-Cre) demonstrate significantly reduced exercise time to exhaustion and decreased running distance compared to control mice by 9 months of age .
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Administration of recombinant CREG1 protein can improve motor function in creg1;Ckm-Cre mice, suggesting therapeutic potential .
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At the ultrastructural level, electron microscopy reveals abnormal mitochondrial quality and quantity in the skeletal muscles of 9-month-old creg1;Ckm-Cre mice .
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Molecularly, CREG1 deficiency is associated with increased levels of mitophagy regulators PINK1 and PARKIN, and reduced levels of mitochondrial proteins PTGS2/COX2, COX4I1/COX4, and TOMM20 .
These findings indicate that CREG1 plays a critical role in maintaining mitochondrial homeostasis and function in skeletal muscle during aging.
What is CREG1's role in cardiac function, particularly in diabetic cardiomyopathy?
CREG1 serves as a protective factor against diabetic cardiomyopathy through several mechanisms:
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In a type 2 diabetes mouse model, CREG1 deficiency (using Creg1-CKO mice) exacerbates cardiac dysfunction, cardiac hypertrophy, and fibrosis .
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Conversely, CREG1 overexpression (using Creg1-TG mice) improves cardiac function and reduces cardiac hypertrophy and fibrosis in diabetic cardiomyopathy .
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The protective effects of CREG1 are mediated through improved autophagy in cardiomyocytes .
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CREG1 operates through a CREG1-FBXO27-LAMP2 axis to regulate autophagy in the context of diabetic cardiomyopathy .
These findings establish CREG1 as an important regulator of cardiac function under diabetic stress conditions, primarily through its effects on autophagy.
How does CREG1 influence age-related metabolic phenotypes?
CREG1 has significant effects on age-related metabolic parameters:
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aP2-CREG1-Tg mice (with enhanced CREG1 expression in adipose tissues) show resistance to age-associated weight gain compared to wild-type mice .
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Age-related increases in blood glucose levels are suppressed in CREG1 transgenic mice .
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Total blood cholesterol concentration is decreased in aged CREG1 transgenic mice at 15 and 18 months of age .
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CREG1 overexpression is associated with increased brown fat formation in aged mice, which may contribute to improved metabolic profiles .
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CREG1 heterozygous mice are susceptible to diet-induced obesity and insulin resistance, further supporting CREG1's protective role in metabolism .
These findings position CREG1 as a potential therapeutic target for age-related metabolic disorders.
What cell culture models are effective for studying CREG1 function?
Several cell culture models have been validated for CREG1 research:
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Neonatal mouse cardiomyocytes (NMCMs): Isolated from 1-3 day-old mice using primary cardiomyocyte isolation kits. These cells allow investigation of CREG1's role in cardiomyocyte function and can be manipulated with:
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C2C12 myoblasts: Useful for studying CREG1's role in skeletal muscle cells, particularly with respect to mitochondrial function and mitophagy .
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H9C2 cells: Cardiac-derived cell line suitable for protein interaction studies using immunoprecipitation approaches .
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3T3 fibroblasts: Used for protein interaction studies through mass spectrometry analysis of CREG1-interacting partners .
For all models, genetic manipulation approaches include:
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siRNA transfection (e.g., targeting sequence GCCACTATCTCCACAATAA for CREG1)
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Adenoviral transduction for overexpression
What techniques are essential for assessing CREG1 expression and function?
A comprehensive analysis of CREG1 requires multiple complementary techniques:
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Protein expression analysis:
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mRNA expression analysis:
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Protein interaction studies:
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Functional assays:
How should researchers design experimental protocols to study age-dependent changes in CREG1 expression?
When investigating age-dependent changes in CREG1 expression, consider the following methodological approaches:
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Age selection: Include multiple age points that span young (3-5 months), middle-aged (9-15 months), and old (18-25 months) mice to capture the complete trajectory of age-related changes .
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Tissue selection: Analyze multiple tissues, as age-related changes in CREG1 expression vary significantly between tissues:
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Expression analysis: Measure both mRNA and protein levels, as there may be post-transcriptional regulation:
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Sample size calculation: Use power analysis based on preliminary data to determine appropriate sample sizes for detecting age-related differences, typically 6-8 mice per group .
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Control considerations: Include both age-matched and genotype-matched controls when studying transgenic models .
The data from source reveal that wild-type mice show a significant age-related increase in serum CREG1 levels (3.64-fold increase at 24 months compared to 9 months), while this age-related increase is not observed in CREG1 transgenic mice. This suggests complex regulatory mechanisms that should be considered in experimental design.
How does CREG1 regulate mitochondrial function and mitophagy?
CREG1 plays a critical role in mitochondrial homeostasis through several mechanisms:
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Mitochondrial localization: CREG1 has been demonstrated to localize to mitochondria, positioning it to directly influence mitochondrial function .
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Mitophagy regulation: CREG1 deficiency accelerates mitophagy in skeletal muscle, as evidenced by:
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Protein interactions: CREG1 (130-220 aa) interacts with HSPD1/HSP60 (401-573 aa), which antagonizes CREG1 degradation and is involved in mitophagy regulation .
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Functional consequences: The mitochondrial regulatory role of CREG1 has physiological implications, as evidenced by reduced exercise capacity in muscle-specific knockout mice and its improvement with recombinant CREG1 administration .
These findings establish CREG1 as an important regulator of mitochondrial quality control, with implications for muscular diseases and aging.
What molecular partners interact with CREG1 to mediate its functions?
CREG1 interacts with several protein partners that mediate its diverse cellular functions:
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HSPD1/HSP60 (Heat shock protein 1):
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FBXO27 (F-box protein 27):
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LAMP2 (Lysosomal-associated membrane protein 2):
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FBXO6 (F-box protein 6):
These protein interactions can be studied using techniques like immunoprecipitation followed by western blotting or mass spectrometry, as well as co-localization studies using immunofluorescence microscopy .
How do tissue-specific and age-dependent changes in CREG1 expression reconcile with its diverse physiological roles?
The relationship between CREG1 expression patterns and its physiological roles presents a complex research challenge:
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Tissue-specific expression patterns:
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Age-dependent expression changes:
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In wild-type mice, serum CREG1 levels increase dramatically with age (3.64-fold increase at 24 months compared to 9 months)
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Liver CREG1 expression increases 1.4-fold in aged wild-type mice compared to young mice
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Kidney CREG1 expression increases 3.5-fold in aged wild-type kidneys compared to young kidneys
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These age-related increases are not observed in CREG1 transgenic mice
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Reconciling diverse physiological roles:
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In skeletal muscle: Maintains mitochondrial homeostasis and exercise capacity
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In heart: Protects against diabetic cardiomyopathy through autophagy regulation
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In metabolism: Reduces age-related weight gain and blood glucose increases
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In kidney: Alleviates age-related morphological abnormalities and improves filtering function
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The apparent paradox of increased CREG1 levels with age despite age-related decline in tissue function suggests complex regulatory mechanisms that may involve compensatory upregulation in response to cellular stress or altered post-translational modifications affecting CREG1 activity.
Future research should explore whether tissue-specific post-translational modifications of CREG1 occur with aging and how these modifications might affect its function in different physiological contexts.
What methodological challenges exist in studying the role of CREG1 in multiple physiological systems?
Researchers face several methodological challenges when investigating CREG1:
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Distinguishing local vs. systemic effects:
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Temporal considerations:
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Molecular mechanism diversity:
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Compensatory mechanisms:
These challenges necessitate careful experimental design with appropriate controls and complementary approaches to fully understand CREG1's multifaceted roles.
How can apparent contradictions in CREG1 research findings be reconciled?
Several apparent contradictions exist in the CREG1 research literature that require careful consideration:
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Age-related expression patterns:
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Contradiction: Wild-type mice show increased CREG1 levels with age, yet aging is associated with deteriorating tissue function that CREG1 appears to protect against .
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Reconciliation approach: Investigate whether age-related CREG1 upregulation represents a compensatory mechanism that is ultimately insufficient to prevent age-related decline. Also examine potential age-related changes in CREG1 post-translational modifications that might alter its activity.
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Transgenic expression effects:
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Contradiction: CREG1 transgenic mice show higher CREG1 levels at young ages but lower levels than wild-type mice at older ages .
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Reconciliation approach: Explore negative feedback mechanisms that might regulate endogenous CREG1 expression in response to transgenic overexpression. Investigate whether transgenic expression alters CREG1 stability or clearance.
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Tissue-specific functions:
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Contradiction: CREG1 regulates mitophagy in skeletal muscle but general autophagy in cardiac tissue .
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Reconciliation approach: Determine whether tissue-specific protein interactions direct CREG1 toward different autophagic pathways. Compare CREG1 interactomes between tissues using immunoprecipitation followed by mass spectrometry.
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These contradictions highlight the complexity of CREG1 biology and emphasize the need for integrative approaches that consider tissue specificity, developmental timing, and molecular context.
Product Science Overview
CREG1 is implicated in maintaining cellular homeostasis and has been shown to play a role in various physiological and pathological processes:
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Tumor Suppression: CREG1 has been identified as a tumor suppressor. It is negatively regulated by cathepsin proteases, particularly cathepsin B. Over-expression of cathepsin B reduces the abundance of CREG1, while inhibition or deletion of cathepsin B increases its levels . This regulation suggests that CREG1 plays a crucial role in tumor-stroma interactions and malignant cell behavior.
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Vascular Remodeling: CREG1 is a critical determinant of vascular remodeling in response to angiotensin II (Ang II). It has been shown that CREG gene expression is significantly decreased in remodeled vascular tissues of high salt-induced Dahl salt-sensitive rats and Ang II-induced mice . This downregulation is Ang II specific and independent of blood pressure. CREG1’s role in vascular remodeling highlights its potential as a therapeutic target for preventing vascular diseases.
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Cardiac Hypertrophy: CREG1 has been shown to attenuate cardiac hypertrophy in vitro. Although its role in vivo has not been fully determined, it is suggested that CREG1 could be beneficial in reducing cardiac hypertrophy .
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Cellular Senescence: CREG1 accelerates p16-dependent cellular senescence in vitro. It also stimulates brown adipogenesis, as observed in adipocyte P2-CREG1-transgenic (Tg) mice . This indicates that CREG1 may have a role in aging-associated phenotypes and metabolic processes.
Research on CREG1 continues to uncover its multifaceted roles in cellular processes. The recombinant form of CREG1, particularly in mouse models, has been instrumental in studying its functions and potential therapeutic applications. For instance, recombinant CREG1 has been used to reduce proliferation, migration, and invasion of tumor cells in vitro and in vivo .
In conclusion, Cellular Repressor of E1A-Stimulated Genes 1 (Mouse Recombinant) is a vital protein with significant implications in tumor suppression, vascular remodeling, cardiac hypertrophy, and cellular senescence. Its diverse roles make it a promising target for therapeutic interventions in various diseases.
