Recombinant Mouse Protein FAM168B (Fam168b)

What is FAM168B protein and what are its known biological functions?

FAM168B (Family with sequence similarity 168 member B) is a protein also known as Myelin-associated neurite-outgrowth inhibitor (MANI) . Current research indicates its importance in central nervous system development, particularly in myelin formation. In zebrafish models, FAM168B has been shown to be predominantly expressed in the brain by 96 hours post-fertilization (hpf), with expression patterns similar to pou3f1, another neural development factor .

Functionally, studies reveal that knockdown or knockout of fam168b results in developmental defects including shortened body, microcephalia, and slight ventralization . These phenotypes suggest critical roles in neural development across vertebrate species.

What are the structural properties of recombinant mouse FAM168B protein?

Recombinant mouse FAM168B is characterized as follows:

The human variant has a calculated molecular weight of approximately 20 kDa based on its 195 amino acid sequence . The mouse protein is likely of similar size, though exact molecular weight specifications were not provided in the available data .

How does FAM168B expression change during embryonic development?

This temporal and spatial regulation suggests stage-specific functions that correlate with neural tissue differentiation and the onset of myelination processes. The expression pattern at 96 hpf resembles that of pou3f1, another transcription factor involved in neural development, indicating potential functional relationships or shared regulatory networks .

What are the optimal conditions for storing and reconstituting recombinant FAM168B protein?

For optimal results when working with recombinant FAM168B protein, researchers should follow these storage and reconstitution guidelines:

Repeated freezing and thawing is not recommended as it may compromise protein integrity .

What detection methods and antibodies are available for studying FAM168B in tissue samples?

For immunohistochemical detection of FAM168B in tissue samples, polyclonal antibodies are available with the following specifications:

The antibody targets FAM168B in IHC applications and has been validated with human samples . While not explicitly mentioned in the available data, antibodies described for IHC may potentially be used for Western blot detection of FAM168B in protein extracts, though optimization would be necessary.

What expression systems are most effective for producing recombinant mouse FAM168B?

E. coli has been successfully used as an expression system for recombinant full-length mouse FAM168B protein . The protein can be expressed with an N-terminal His tag to facilitate purification using affinity chromatography. This approach typically yields protein with greater than 90% purity as determined by SDS-PAGE .

While the available information specifically documents E. coli expression, other systems such as mammalian, insect, or yeast cells might provide alternatives with different post-translational modifications or folding characteristics, though these would need to be empirically evaluated.

What is the relationship between FAM168B and central nervous system myelin formation?

FAM168B plays a significant role in central nervous system (CNS) myelin formation. Research in zebrafish models has demonstrated that:

• Knockdown or knockout of fam168b results in reduced expression of myelin basic protein (mbp) and proteolipid protein 1a (plp1a), which are key markers of myelin formation .

• In copper-stressed zebrafish embryos, which exhibit myelin defects, fam168b expression is significantly reduced .

• When fam168b mRNA is injected into copper-stressed embryos, partial recovery of mbp expression is observed, suggesting a rescue of myelin formation .

These findings collectively indicate that FAM168B is required for proper myelination in the CNS and that its expression levels directly impact myelin marker expression and formation.

How does FAM168B interact with other factors in neural development pathways?

FAM168B functions within a complex regulatory network involving several other factors critical for neural development:

• FAM168A: Shows functional overlap with FAM168B. Knockdown of either fam168a or fam168b results in similar developmental defects. Transcriptional profiling reveals that 104 genes in the nervous system and 8 genes in synapse formation are down-regulated in both fam168a and fam168b morphants .

• POU3F1: A transcription factor with expression patterns similar to FAM168B. Morphants for fam168b and pou3f1 show overlapping transcriptional changes, with 85 genes in the nervous system and 8 genes in synapse formation down-regulated in all three fam168a, fam168b, and pou3f1 morphants .

• HOXB5B: Part of the Wnt and Notch signaling pathways. A complex regulatory relationship exists, as fam168b expression significantly increases in both hoxb5b morphants and hoxb5b-/- mutants, while hoxb5b expression increases in fam168b morphants .

This interconnected regulatory network underscores the complexity of neural development pathways and positions FAM168B as an important component in these processes.

How does the promoter methylation of FAM168B affect its expression and function?

Research has revealed important insights about FAM168B promoter methylation:

• Different hypermethylated loci in the fam168b promoter, including regions from -1672 to -1414, -1414 to -1240, and -1240 to -927, are critical for its transcriptional regulation .

• Deletion of these loci induces significant down-regulation of transcriptional activity, suggesting these hypermethylated regions are required for fam168b transcriptional activation .

• In copper-stressed zebrafish embryos, copper induces hypermethylation in the fam168b promoter, which correlates with down-regulated gene expression .

Interestingly, the temporal dynamics show that down-regulated expression of fam168b occurs at 24 hpf, followed by hypermethylation of its promoter at 96 hpf in copper-stressed embryos. This suggests that copper might initially damage the chromatin structure of the transcriptional complex before promoter hypermethylation occurs .

What are the implications of copper-induced FAM168B dysregulation for neurodevelopmental studies?

Copper exposure significantly impacts FAM168B expression and function, with important implications for neurodevelopmental research:

• In zebrafish embryos exposed to copper (Cu²⁺), fam168b expression is notably reduced, coinciding with central nervous system myelin defects .

• The mechanism involves hypermethylation of specific loci in the fam168b promoter that are critical for transcriptional regulation .

• This copper-induced dysregulation provides a model for how environmental toxins might contribute to developmental disorders through epigenetic mechanisms .

These findings suggest FAM168B could serve as a molecular marker for assessing neurodevelopmental toxicity of environmental contaminants, particularly copper. The research also highlights potential epigenetic mechanisms through which environmental exposures might affect neural development and myelination processes.

What approaches are most effective for studying loss-of-function effects of FAM168B?

Based on current research methods, several approaches have proven effective for studying FAM168B loss-of-function:

• Morpholino-mediated knockdown: Injection of morpholinos targeting FAM168B mRNA effectively reduces its expression during embryonic development, allowing assessment of developmental impacts .

• CRISPR/Cas9-mediated knockout: Generation of fam168b-/- mutants with specific deletions (e.g., 1-bp deletion) provides a model for studying complete loss of function .

• Combined knockdown approaches: Given the potential functional redundancy between FAM168A and FAM168B, studies have employed combined knockdowns using morpholinos against both genes to more fully reveal their functions .

• Assessment methods should include:

  • Morphological analysis (body length, head size)

  • Myelin marker expression (mbp, plp1a)

  • Transcriptional profiling to identify affected pathways

• Rescue experiments: Injection of FAM168B mRNA into knockdown or knockout models can confirm specificity of observed phenotypes .

What experimental designs best address potential compensatory mechanisms between FAM168A and FAM168B?

When investigating the potentially overlapping functions of FAM168A and FAM168B, researchers should consider these experimental approaches:

• Individual and combined knockdowns/knockouts: Compare phenotypes from fam168a knockdown, fam168b knockdown, and combined fam168a+fam168b knockdown to identify unique and shared functions .

• Expression analysis: Measure fam168a expression in fam168b knockouts and vice versa to quantify compensatory upregulation.

• Heterozygous mutant combinations: Research has shown that hoxb5b+/-fam168a+/- mutants exhibit normal-like phenotypes at 96 hpf but demonstrate reduced expression of CNS myelin marker mbp, indicating the importance of gene dosage effects .

• Transcriptional profiling: Compare the transcriptomic changes in individual versus combined knockdowns. Current research shows 104 genes in the nervous system and 8 genes in synapse formation are down-regulated in both fam168a and fam168b morphants, indicating substantial functional overlap .

• Rescue experiments: Test whether fam168a overexpression can rescue fam168b loss-of-function phenotypes and vice versa.

How might FAM168B function in neurological disorders beyond development?

While current research focuses predominantly on FAM168B's developmental roles, its function as a myelin-associated neurite-outgrowth inhibitor suggests potential implications for various neurological conditions:

• Demyelinating disorders: Given its role in myelin formation, FAM168B might be involved in demyelinating conditions or in remyelination processes following injury .

• Neurodegenerative diseases: The protein's involvement in myelin maintenance could be relevant to neurodegenerative conditions with white matter pathology.

• Traumatic brain injury: As a neurite-outgrowth inhibitor, FAM168B might influence axonal regeneration following trauma .

Research investigating FAM168B expression and function in these contexts could reveal new therapeutic targets or biomarkers.

What are the most promising approaches for translating FAM168B research to therapeutic applications?

Based on current understanding of FAM168B, several translational research directions appear promising:

• Epigenetic modulation: Given the regulatory role of promoter methylation on FAM168B expression, compounds that modulate this epigenetic mechanism might offer therapeutic potential in conditions with FAM168B dysregulation .

• Targeted delivery systems: For conditions requiring FAM168B supplementation, recombinant protein or mRNA delivery specifically to affected neural tissues could be explored.

• High-throughput screening: Identification of small molecules that modulate FAM168B expression or function could yield potential therapeutic candidates.

• Gene therapy approaches: For genetic conditions affecting FAM168B expression, CRISPR-based or viral vector approaches might enable gene correction or supplementation.

• Biomarker development: FAM168B expression levels or promoter methylation status could potentially serve as biomarkers for neurodevelopmental disorders or environmental toxin exposure .

Recombinant Mouse FAM168B Protein Specifications

FAM168B Antibody Specifications for Research Applications