FGF 1 Mouse

What is FGF1 and what are its primary functions in mouse models?

FGF1, also known as acidic FGF or HBGF-1, is a 17 kDa non-glycosylated member of the FGF family that regulates numerous biological and physiological processes in mice. It functions as a signaling molecule involved in cell proliferation, differentiation, and tissue repair. In mouse models, FGF1 has been extensively studied for its roles in cardiac function, adipose tissue homeostasis, and metabolic regulation. The protein is expressed in multiple tissues with particularly notable expression in the heart and kidneys . FGF1 induces proliferation in mouse fibroblast cell lines such as NR6R-3T3 in a dose-dependent manner, which can be neutralized using specific antibodies, providing a useful bioassay for functional studies .

In transgenic mouse models, FGF1 has demonstrated significant capacity to stimulate cardiomyocyte cell cycle reentry through the FGFR1/Fn14 pathway and regulate cardiomyocyte differentiation via the FGF1/FGFR/PKC signaling axis. Furthermore, it shows therapeutic potential in cardiac injury models by inducing cardiomyocyte regeneration and improving function after myocardial infarction .

How is the Fgf1 gene structured in mice and what transcript variants exist?

In mice, the Fgf1 gene comprises at least three tissue-specific promoters that generate three alternatively spliced transcripts: Fgf1A, Fgf1B, and Fgf1G. These variants differ in their 5' untranslated regions but encode the same protein, as they all splice to the first protein-coding exon . The transcript variants show distinct tissue-specific expression patterns:

• Fgf1A: Predominantly expressed in the heart and kidney under normal conditions; also expressed in adipose tissue in obesity models

• Fgf1B: Shows different tissue distribution compared to Fgf1A

• Fgf1G: Expressed in specific tissues distinct from Fgf1A and Fgf1B

The mouse Fgf1A transcript includes a 5' UTR of approximately 127.7 kb, followed by exon 1A, which is critical for tissue-specific expression . This complex gene structure allows for precise regulation of FGF1 expression in different physiological contexts.

What detection methods are available for studying FGF1 in mouse tissues?

Several complementary methods can be employed to detect and quantify FGF1 in mouse tissues:

• Western Blot Analysis: Using specific antibodies such as Sheep Anti-Mouse FGF acidic/FGF1 Antigen Affinity-purified Polyclonal Antibody. Western blot can detect FGF1 in tissue lysates (e.g., heart and kidney tissues) as a specific band of approximately 18 kDa under reducing conditions .

• Quantitative PCR (qPCR): For measuring Fgf1 transcript levels in different tissues. This approach can distinguish between different Fgf1 transcript variants using specific primers .

• Immunohistochemistry/Immunofluorescence: For in situ detection of FGF1 protein in tissue sections, often combined with markers for specific cell types (e.g., cardiac troponin T for cardiomyocytes) .

• Reporter Systems: Transgenic mice expressing reporters (LacZ, RFP) under the control of Fgf1 promoters enable visualization of Fgf1 expression patterns .

• Cell Proliferation Assays: Functional assays using NR6R-3T3 mouse fibroblasts can measure FGF1 bioactivity. The neutralization dose (ND50) is typically 0.1-0.6 μg/mL in the presence of 1 ng/mL recombinant mouse FGF1 .

What are the recommended storage conditions for mouse FGF1 reagents?

For optimal stability and activity of mouse FGF1 reagents, the following storage conditions are recommended:

• Long-term storage: Use a manual defrost freezer at -20 to -70 °C for up to 12 months from date of receipt for unopened reagents

• After reconstitution under sterile conditions:

  • 1 month at 2 to 8 °C

  • 6 months at -20 to -70 °C

It is crucial to avoid repeated freeze-thaw cycles as these can significantly reduce protein activity. Aliquoting reconstituted protein into single-use volumes is recommended for research requiring multiple experiments over time .

How can the F1A-CreERT2 mouse line be used for lineage tracing of Fgf1-expressing cells?

The F1A-CreERT2 transgenic mouse line provides a powerful tool for time-dependent and lineage tracing of Fgf1A-expressing cells in vivo. This model was generated using a BAC clone containing the Fgf1A 5'UTR (127.7 kb) and exon 1A sequence, with an RFP-IRES-CreERT2-polyA cassette inserted into exon 1A through homologous recombination .

Experimental methodology for lineage tracing:

• Cross F1A-CreERT2 mice with reporter strains: The F1A-CreERT2 mice should be crossed with reporter mice such as ROSA26 LacZ reporter mice to generate F1 mice carrying both transgenes .

• Tamoxifen administration: Inject tamoxifen to activate the CreERT2 recombinase at specific time points. This induces recombination specifically in cells where the Fgf1A promoter is active at the time of injection .

• Analysis of reporter expression:

  • For LacZ reporters: Perform β-galactosidase staining on tissue sections

  • For fluorescent reporters: Direct visualization of fluorescence (e.g., RFP) in tissue sections

• Co-localization studies: Combine reporter detection with immunostaining for cell-type specific markers (e.g., cardiac troponin T for cardiomyocytes) to identify the specific cell populations expressing Fgf1A .

The F1A-CreERT2 mouse model has revealed that in adult mice, Fgf1A promoter activity is predominantly found in cardiomyocytes, with reporter signals detected in various heart regions including right atrium (RA), right ventricle (RV), atrioventricular node (AV), interventricular septum (IVS), left atrium (LA), and left ventricle (LV) .

What is the role of FGF1 in cardiomyocyte regeneration and what experimental models best study this process?

FGF1 plays significant roles in cardiomyocyte regeneration and cardiac repair through several mechanisms. Research has demonstrated that FGF1 can stimulate neonatal cardiomyocyte cell cycle reentry through the FGFR1/Fn14 pathway, regulate cardiomyocyte differentiation from embryonic stem cells via the FGF1/FGFR/PKC signaling axis, and induce cardiomyocyte regeneration after myocardial infarction .

Experimental models for studying FGF1 in cardiac regeneration:

• In vitro models:

  • Neonatal cardiomyocyte cultures: Used to study cell cycle reentry induced by FGF1

  • Embryonic stem cell differentiation: To investigate FGF1's role in cardiomyogenesis

  • NR6R-3T3 mouse fibroblast cell line: For proliferation assays to assess FGF1 activity

• In vivo models:

  • Myocardial infarction models: To study FGF1's effect on cardiac repair and regeneration

  • Diabetic cardiomyopathy models: To investigate FGF1's protective effects against oxidative stress

  • F1A-CreERT2 transgenic mice: For lineage tracing of Fgf1-expressing cells during cardiac regeneration

• Combination approaches:

  • FGF1/neurogenin1 co-administration: Shown to stimulate cardiomyocyte proliferation and facilitate cardiac remodeling after myocardial infarction

  • Conditional knockout or overexpression models: To investigate tissue-specific and temporal requirements for FGF1 signaling

The F1A-CreERT2 mouse line provides a valuable tool for these studies by allowing researchers to track the fate of Fgf1-expressing cells during cardiac development, injury, and regeneration processes .

How does FGF1 contribute to metabolic regulation in mouse models of diabetes and obesity?

FGF1 plays crucial roles in metabolic regulation, particularly in glucose homeostasis and adipose tissue function. In mouse models of diabetes and obesity, several key findings have emerged:

• Adipose tissue homeostasis: FGF1 regulates adipose tissue homeostasis, with Fgf1 knockout mice showing an aggressive diabetic phenotype when fed a high-fat diet, indicating its importance in metabolic adaptation .

• Glucose regulation: Subcutaneous injection of FGF1 protein significantly improves insulin sensitivity and reduces serum glucose levels in multiple mouse models of diabetes:

  • ob/ob mice (leptin deficiency)

  • db/db mice (leptin receptor deficiency)

  • Diet-induced obese insulin-resistant mice

• Rapid and sustained effects: Significant reduction in serum glucose levels can be achieved within 24 hours of FGF1 administration and can last more than 48 hours. Importantly, even at higher doses, FGF1 does not induce hypoglycemia in diabetic mice, suggesting a regulated mechanism of action .

• β-cell independent effects: FGF1 can reduce serum glucose levels even in streptozotocin-induced diabetic mice, which have lost β-cells, indicating mechanisms beyond insulin secretion .

• Anti-inflammatory action: Long-term FGF1 administration ameliorates systemic inflammation in ob/ob mice, functioning through the FGFR1 pathway and acting similar to thiazolidinediones as an insulin sensitizer .

Experimental approaches to study FGF1 in metabolism:

• High-fat diet feeding with FGF1 administration

• Glucose tolerance tests and insulin tolerance tests

• Analysis of inflammatory markers

• Adipose tissue-specific gene expression analysis

• Comparison of wild-type and Fgf1 knockout mice under metabolic challenge

What technical considerations are important when generating and characterizing FGF1 transgenic mouse models?

Creating and properly characterizing FGF1 transgenic mouse models requires careful attention to several technical considerations:

• Promoter selection and design:

  • For tissue-specific expression, selecting the appropriate Fgf1 promoter variant (A, B, or G) is crucial

  • The F1A-CreERT2 model used a 127.7 kb 5'UTR region to ensure proper regulation

  • BAC clones provide comprehensive regulatory elements for physiological expression patterns

• Recombination system design:

  • For inducible systems like CreERT2, optimizing expression is critical

  • The F1A-CreERT2 model used the native internal ribosome entry site (IRES) from encephalomyocarditis virus with A6 bifurcation sequence to enhance translation efficiency

• Founder selection and validation:

  • Multiple founder lines should be generated and compared

  • The F1A-CreERT2 model identified three potential founders, but only one (#13A) showed sufficient expression levels for functional studies

  • Quantitative PCR for transgene expression in target tissues is essential for selecting appropriate founders

• Reporter system integration:

  • Including reporter genes (RFP, LacZ) facilitates visualization of expression patterns

  • Bicistronic expression cassettes (e.g., RFP-IRES-CreERT2) allow simultaneous visualization and genetic manipulation

• Validation of expression patterns:

  • Compare transgene expression with endogenous gene expression patterns

  • For F1A-CreERT2 mice, CreERT2 mRNA expression matched endogenous Fgf1A mRNA patterns

  • Validate cell-type specificity through co-localization studies with appropriate markers

• Functional validation:

  • Cross with reporter strains (e.g., ROSA26) to confirm recombinase activity

  • Test induction protocols (e.g., tamoxifen dosing) for optimal temporal control

  • Verify phenotype absence in unmodified conditions to ensure the model doesn't have inherent defects

How can FGF1 activity be quantitatively measured in experimental mouse models?

Quantitative assessment of FGF1 activity in mouse models can be approached through several complementary methods:

• Cell proliferation assays:

  • The NR6R-3T3 mouse fibroblast cell line shows dose-dependent proliferation in response to FGF1

  • Proliferation can be quantified and used as a bioassay for FGF1 activity

  • The response can be neutralized by anti-FGF1 antibodies, with a typical neutralization dose (ND50) of 0.1-0.6 μg/mL in the presence of 1 ng/mL recombinant mouse FGF1

• Western blot quantification:

  • Tissue lysates (e.g., heart, kidney) can be analyzed for FGF1 protein levels

  • FGF1 appears as an approximately 18 kDa band under reducing conditions

  • Quantification should be performed using appropriate loading controls

• Transcriptional analysis:

  • qPCR for Fgf1 mRNA variants and FGF1-responsive genes

  • RNA-seq for comprehensive transcriptional profiling

  • Analysis should distinguish between different Fgf1 transcript variants (1A, 1B, 1G)

• In vivo functional assays:

  • For cardiac studies: echocardiography, pressure-volume measurements

  • For metabolic studies: glucose tolerance tests, insulin sensitivity tests

  • For regeneration studies: BrdU or EdU incorporation to measure proliferation

• Reporter systems:

  • In F1A-CreERT2 x ROSA26 mice, β-galactosidase activity (LacZ staining) provides a visual and quantifiable readout of cells with active Fgf1A promoter

  • Direct visualization of RFP signals in F1A-CreERT2 mice allows for quantification of reporter-positive cells

• ELISA and immunoassays:

  • For measuring circulating FGF1 levels in serum or plasma

  • For quantifying FGF1 in tissue homogenates or culture supernatants

These methods can be combined to provide comprehensive assessment of FGF1 activity at molecular, cellular, and physiological levels in experimental mouse models.

What are the optimal tamoxifen administration protocols for F1A-CreERT2 mice?

For effective temporal control of Cre-mediated recombination in F1A-CreERT2 mice, proper tamoxifen administration is crucial. While the provided search results don't specify exact protocols for this particular mouse line, general considerations based on standard practices for CreERT2 systems include:

• Delivery methods:

  • Intraperitoneal (IP) injection: Most common method for adult mice

  • Oral gavage: Alternative to IP injections

  • Tamoxifen-containing food: For more gradual, chronic induction

• Dosage considerations:

  • For adult mice, 1-2 mg tamoxifen per 25g body weight is typically used

  • Multiple injections (3-5 consecutive days) are often needed for optimal recombination

  • Dose finding studies should be performed to determine optimal conditions for each specific CreERT2 line

• Timing for lineage tracing:

  • Allow 24-72 hours after the last tamoxifen dose before analyzing acute effects

  • For long-term lineage tracing, reporter expression becomes more robust 5-7 days after induction

• Preparation of tamoxifen:

  • Dissolve in corn oil or sunflower oil at concentrations of 10-20 mg/mL

  • For complete dissolution, sonication and heating (37°C) may be required

  • Prepare fresh solutions or store at -20°C protected from light for limited periods

• Controls:

  • Oil-only injections in F1A-CreERT2 x reporter mice

  • Tamoxifen injections in reporter-only mice

  • These controls are essential to rule out leaky Cre activity or tamoxifen-related effects

In the F1A-CreERT2 mice, tamoxifen administration resulted in CreERT2-mediated recombination specifically in cardiomyocytes, with no recombination observed in other cell types, confirming the cardiomyocyte-specific activation of the Fgf1A promoter .

How should tissue preparation be optimized for detecting FGF1 and reporter signals in mouse tissues?

Proper tissue preparation is critical for reliable detection of FGF1 protein and reporter signals in transgenic mouse models. Based on the provided research, the following protocols have proven effective:

• For direct fluorescence detection (e.g., RFP):

  • Anesthetize mice with isoflurane

  • Perform intracardiac perfusion with ice-cold PBS without calcium and magnesium

  • Fix harvested tissues in pre-chilled 95% ethanol for 20-24h at 4°C

  • Dehydrate in 4 changes of pre-chilled 100% ethanol for 1h at 4°C

  • Embed in optimal cutting temperature (OCT) compound

  • Store at -70°C

  • Cryosection to 6-μm thickness

  • Rinse with water and observe directly under a fluorescent microscope without additional staining

• For LacZ staining:

  • Following appropriate tamoxifen administration in F1A-CreERT2 x ROSA26 mice

  • Harvest tissues and process according to standard β-galactosidase staining protocols

  • This allows visualization of cells where Cre-mediated recombination has occurred

• For immunohistochemical detection:

  • Use appropriate fixation based on antibody requirements (typically 4% paraformaldehyde)

  • For co-localization studies with cell type markers (e.g., cardiac troponin T), optimize fixation to preserve epitopes for all antibodies

  • When detecting both RFP signals and immunofluorescence staining, careful optimization of fixation is crucial to maintain RFP fluorescence while allowing antibody binding

• For Western blot analysis:

  • Prepare tissue lysates under reducing conditions

  • Probe membranes with 2 μg/mL of anti-FGF1 antibody followed by appropriate secondary antibody

  • FGF1 is detected as a specific band at approximately 18 kDa

These protocols can be adapted based on specific experimental requirements and should be optimized for each application and tissue type.

How can researchers address weak or variable FGF1 expression in mouse models?

When facing challenges with weak or variable FGF1 expression in mouse models, researchers should consider several troubleshooting approaches:

• Founder line selection:

  • As demonstrated with the F1A-CreERT2 mice, significant variation can exist between founder lines

  • In the reported study, strain #13A showed the highest gene expression levels, with Ct value differences of 8.5-9.3 compared to other strains

  • Always generate and screen multiple founder lines when creating new transgenic models

• Tissue collection and processing optimization:

  • Use rapid extraction and appropriate preservation methods

  • For RNA analysis, use RNase inhibitors and quick freezing

  • For protein analysis, include protease inhibitors in all buffers

• Age and sex considerations:

  • FGF1 expression may vary with age and sex

  • Standardize experimental groups by age and include both sexes or justify single-sex studies

  • Consider developmental timing for embryonic or neonatal studies

• Environmental and physiological factors:

  • Feeding status, time of day, stress levels, and housing conditions can affect gene expression

  • Standardize conditions and collection times

  • Record any potential confounding variables

• Detection method sensitivity:

  • For low expression, consider more sensitive methods (e.g., droplet digital PCR instead of qPCR)

  • Use amplification steps in immunohistochemistry protocols

  • Consider concentration steps for protein detection

• Genetic background effects:

  • Backcross to a consistent genetic background for more reliable results

  • Be aware that modifier genes can influence FGF1 expression and function

• Induction protocol optimization:

  • For inducible systems like F1A-CreERT2, optimize tamoxifen dosing and timing

  • Consider dose-response studies to determine optimal conditions

What controls are essential when studying FGF1 function in transgenic mouse models?

Proper experimental controls are critical for reliable interpretation of results when studying FGF1 function. Essential controls include:

• Genetic controls:

  • Wild-type littermates: Essential baseline control

  • Heterozygous vs. homozygous comparisons: To assess gene dosage effects

  • Cre-only and floxed-only controls: For conditional systems to rule out effects of Cre expression or loxP sites

• Induction controls for CreERT2 systems:

  • Vehicle-only administration (e.g., oil without tamoxifen)

  • Reporter-only mice with tamoxifen: To check for tamoxifen-specific effects

  • Temporal controls: Different time points after induction to track dynamic changes

• Tissue specificity controls:

  • Multiple tissue analysis: Verify expected tissue-specific patterns

  • Cell-type markers: Co-staining with markers like cardiac troponin T (for cardiomyocytes) confirms cell type specificity

  • Analysis of tissues expected to be negative for expression

• Functional assay controls:

  • For cell proliferation assays: Include neutralizing antibodies (ND50 typically 0.1-0.6 μg/mL) to confirm specificity of FGF1 effects

  • Dose-response studies: Establish proper dose ranges for observed effects

  • Pathway inhibitors: To confirm suspected signaling mechanisms

• Technical controls:

  • For Western blots: Loading controls, negative control tissues, recombinant protein standards

  • For PCR: No-template controls, reverse transcriptase negative controls

  • For immunostaining: Primary antibody omission, isotype controls, blocking peptide controls

• Physiological controls:

  • Age-matched and sex-matched animals

  • Controlled environmental conditions (diet, housing, handling)

  • Consistent time of day for measurements of physiologically variable parameters

These controls help distinguish specific FGF1-related effects from background variation, technical artifacts, and non-specific responses to experimental manipulations.

What are promising therapeutic applications of FGF1 research in mouse models?

Based on findings from mouse models, FGF1 shows therapeutic potential in several disease areas that warrant further investigation:

• Cardiac regeneration and repair:

  • FGF1 induces cardiomyocyte regeneration and improves cardiac function after myocardial infarction

  • The combination of FGF1 with neurogenin1 stimulates cardiomyocyte proliferation and facilitates cardiac remodeling

  • Future research could optimize delivery methods, timing, and combination therapies for maximal regenerative effect

• Diabetes and metabolic disorders:

  • FGF1 significantly improves insulin sensitivity and reduces serum glucose levels in multiple mouse models of diabetes

  • Unlike insulin, higher doses of FGF1 do not cause hypoglycemia, suggesting a regulated mechanism

  • FGF1 administration ameliorates systemic inflammation in obese mice

  • Future studies could focus on developing FGF1 variants with enhanced stability or receptor specificity for metabolic applications

• Diabetic cardiomyopathy:

  • FGF1 prevents diabetic cardiomyopathy through reduction of oxidative stress

  • The intersection of FGF1's cardiac and metabolic effects makes it particularly promising for complications of diabetes

  • Research into tissue-specific delivery or activation could minimize off-target effects

• Adipose tissue homeostasis:

  • FGF1 regulates adipose tissue homeostasis, with potential applications in obesity

  • Future research could explore adipose depot-specific effects and mechanisms

  • Understanding the crosstalk between FGF1 and adipokines could reveal new therapeutic targets

• Developmental biology applications:

  • The F1A-CreERT2 mouse model enables precise temporal control for developmental studies

  • Future research could use this system to dissect the role of FGF1A-expressing cells during critical developmental windows

  • This could inform regenerative medicine approaches that recapitulate developmental processes

How might single-cell technologies advance FGF1 research in mouse models?

Single-cell technologies offer powerful new approaches to understand FGF1 biology at unprecedented resolution:

• Single-cell RNA sequencing (scRNA-seq):

  • Can identify specific cell populations expressing Fgf1 and its receptors

  • Would reveal heterogeneity within seemingly uniform tissues

  • Could track transcriptional changes in FGF1-responsive cells during development, homeostasis, and disease

  • Would be particularly valuable for heart studies, where the F1A-CreERT2 model has shown cardiomyocyte-specific expression

• Single-cell ATAC-seq:

  • Would reveal chromatin accessibility changes associated with FGF1 signaling

  • Could identify tissue-specific enhancers controlling Fgf1 expression

  • Would complement the promoter studies that identified tissue-specific transcripts (1A, 1B, 1G)

• Spatial transcriptomics:

  • Could map Fgf1 expression in intact tissues with spatial context

  • Would reveal relationships between Fgf1-expressing cells and their neighbors

  • Could be combined with the F1A-CreERT2 model to track spatial dynamics of FGF1-expressing lineages over time

• CRISPR-based lineage tracing:

  • Would enable more sophisticated lineage analysis than traditional Cre-lox systems

  • Could generate cellular barcodes to track clonal dynamics of FGF1-expressing cells

  • Would complement the existing F1A-CreERT2 model with higher resolution lineage information

• Single-cell proteomics and metabolomics:

  • Would reveal how FGF1 signaling affects protein expression and metabolic states at the single-cell level

  • Could identify new downstream effectors of FGF1 signaling

  • Particularly relevant for understanding FGF1's metabolic effects in diabetes models

These technologies could transform our understanding of FGF1 biology by revealing cell-type specific responses, identifying new therapeutic targets, and enabling precision approaches to modulating FGF1 signaling in specific contexts.