FABP3 Human, His

What are the fundamental characteristics of human FABP3 and its His-tagged variants?

Human FABP3, also known as heart-type fatty acid binding protein (H-FABP), is a 15-kDa cytoplasmic protein that facilitates intracellular transport of long-chain fatty acids. The protein's structure includes a β-barrel motif with a central cavity for fatty acid binding. His-tagged variants of FABP3 are recombinant versions containing histidine residues (typically 6×His) that facilitate protein purification through metal affinity chromatography without significantly altering the protein's native function.

When designing experiments with His-tagged FABP3, researchers should consider that human FABP3 cDNA covers the open reading frame and untranslated regions. The gene can be amplified using PCR with specific primer sets as documented in previous studies (FABP3 forward: 5′-CCTGCTCTCTTGTAGCTTCTCTCA-3′, FABP3 reverse: 5′-TGAGGCAATGTGGTGCTGAGTCGA-3′) .

How are FABP3 knockout models generated for research purposes?

FABP3 knockout (F3-KO) models are typically generated using the CRISPR-Cas9 method as demonstrated in recent cardiovascular studies. These models are maintained under standard laboratory conditions (room temperature with a 12-hour light/dark cycle) and are valuable for studying FABP3's biological functions through loss-of-function approaches .

For proper experimental design, researchers should ensure that knockout verification is performed through both genomic analysis and protein expression assessment. Animal procedures should follow appropriate ethical guidelines, such as those from Directive 2010/63/EU or current NIH guidelines, and receive approval from institutional animal care committees .

What molecular techniques are optimal for studying FABP3 variants?

Site-directed mutagenesis is the preferred approach for generating FABP3 variants for functional characterization. This method involves:

• PCR amplification of FABP3 cDNA from appropriate sources (e.g., Marathon-Ready cDNA from human embryonic brain)

• Cloning into expression vectors such as pcDNA3

• Introduction of specific mutations using site-directed mutagenesis

• Verification through DNA sequencing

For expression studies, researchers commonly use mammalian expression vectors (pCMV-HA) for HA-tagged proteins or pAcGFP1-C1 vectors for GFP-tagged proteins. Bacterial expression typically employs pGEX-6P-3 vectors for GST-fusion proteins to facilitate purification .

What behavioral tests are most informative when studying FABP3 function in neurological research?

Based on extensive characterization of Fabp3 knockout mice, several behavioral tests have proven particularly informative:

• Open field test - Particularly valuable for assessing anxiety-like behaviors

• Forced swim test - Shows significant changes in Fabp3-KO models (reduced immobility time, p = 0.0376)

• Prepulse inhibition (PPI) - Demonstrates significant deficits in Fabp3-KO mice

• Social interaction tests - Including the resident-intruder and three-chamber tests

• Pharmacological challenge tests - Such as MK-801 administration to assess NMDA receptor-related behaviors

The following table summarizes key behavioral findings from Fabp3-KO mice compared to other FABP knockouts:

n.s. indicates not significant

How does FABP3 deficiency affect cardiac pathophysiology in hypertrophy models?

FABP3 deficiency significantly exacerbates cardiac hypertrophy in transverse aortic constriction (TAC) models. Key methodological approaches and findings include:

• TAC model induction in 8-week-old mice through aortic arch ligation against a 27-gauge needle

• Echocardiographic assessment following light isoflurane anesthesia

• Analysis of cardiac hypertrophy through:

  • Heart weight to body weight ratio measurements

  • H&E staining of longitudinal heart sections

  • WGA staining to quantify cardiomyocyte size

  • qPCR assays measuring hypertrophic markers (Anp, Bnp, Acta1, Myh7)

Research has shown that FABP3-deficient mice exhibit more severe cardiac hypertrophy after TAC, with significantly increased interventricular septum thickness, left ventricular posterior wall thickness, and heart weight to body weight ratio compared to wild-type controls subjected to the same TAC procedure .

What transcriptomic and metabolomic approaches reveal FABP3's role in cardiac metabolism?

Integrative multi-omics approaches provide comprehensive insights into FABP3's metabolic roles:

• RNA-sequencing (RNA-seq) analysis of heart tissue collected 1-week post-sham or TAC operation

• Liquid chromatography-mass spectrometry (LC-MS) for metabolite profiling

• Principal component analysis (PCA) to assess global transcriptomic differences

• KEGG pathway analysis to identify enriched metabolic pathways

This integrated approach identified 939 differentially expressed genes (772 upregulated, 167 downregulated) when comparing TAC-operated FABP3-knockout hearts with wild-type hearts. Pathway analysis revealed significant enrichment in lipid metabolism, glycan metabolism, and energy metabolism pathways .

How should researchers validate functional consequences of FABP3 variants identified in human disorders?

A comprehensive validation approach should include:

• Site-directed mutagenesis to generate variant constructs in appropriate expression vectors

• Cellular localization studies using fluorescently tagged constructs

• Biochemical assays to assess fatty acid binding capacity

• Functional rescue experiments in knockout models

• In vivo phenotypic characterization using behavioral tests for neurological variants or cardiovascular assessments for cardiac-related variants

When studying variants identified in psychiatric disorders such as schizophrenia or autism spectrum disorder, researchers should complement molecular studies with behavioral characterization in animal models expressing these variants .

What is the optimal experimental design for studying FABP3's role in cardiac hypertrophy?

Based on established protocols, the recommended experimental design includes:

• Generation of FABP3-knockout models using CRISPR-Cas9

• Implementation of the TAC model to induce pathological cardiac hypertrophy:

  • Anesthesia with isoflurane

  • Mechanical ventilation

  • Aortic arch ligation with 6-0 silk suture against a 27-gauge needle

  • Chest closure with 5-0 silk suture

• Comprehensive phenotyping:

  • Echocardiographic analysis

  • Histological assessment (H&E and WGA staining)

  • Molecular analysis (qPCR, Western blotting)

  • Transcriptomic and metabolomic profiling

Control groups should include both wild-type and FABP3-knockout mice subjected to sham operations to differentiate between genotype effects and surgical intervention effects .

How can inconsistencies in FABP3 knockout phenotypes between studies be resolved?

When facing inconsistent results between studies:

• Compare knockout generation methods (traditional homologous recombination vs. CRISPR-Cas9)

• Examine genetic background differences that may influence phenotypes

• Assess environmental factors and housing conditions

• Standardize behavioral testing protocols and analysis methods

• Consider sex differences as a potential variable

• Implement blinded analysis to reduce experimenter bias

The behavioral phenotype of FABP3-knockout mice may vary based on testing conditions, genetic background, and the specific physiological system being assessed. A systematic approach comparing methodologies across studies can help resolve apparent contradictions .

What are the key considerations when designing experiments to study FABP3's role in metabolic pathways?

Critical experimental design considerations include:

• Selection of appropriate metabolic challenge models (e.g., TAC for cardiac studies)

• Integration of multiple analytical approaches:

  • Transcriptomics (RNA-seq)

  • Metabolomics (LC-MS)

  • Functional assays (e.g., fatty acid uptake)

• Temporal analysis to capture dynamic metabolic changes

• Tissue-specific analyses to account for differential expression patterns

• Proper statistical approaches for multi-omics data integration

When studying FABP3's metabolic functions, researchers should design experiments that examine both baseline conditions and stress-induced changes to fully understand the protein's role in homeostatic and pathological states .