In the realm of scientific research, particularly in studies focusing on fat cells and metabolic diseases, accurate and efficient fluorescent detection is essential. This method allows researchers to evaluate recombination efficiency, visualize gene expression profiles, and trace genetic lineages. However, detecting fluorescent signals can sometimes be challenging. If you’ve ever faced difficulties with either targeting adipose tissues or undetectable fluorescence, don’t worry—we’ve got you covered.

Today, we're excited to introduce the Adipoq-iCre Mice (Product No. C001529), a groundbreaking conditional mouse strain designed to specifically target mouse adipose tissues. This innovative model demonstrates adipose-specific gene targeting upon crossing with fluorescent reporter Rosa26-LSL-tdTomato mouse strain, ensuring you can achieve reliable and clear results in your studies. Review the model Immunofluorescence (IF) data herein to see how Adipoq-iCre Mice can be used in adipose-related gene research.

About Adipoq-iCre Mice

Cyagen has independently developed the Adipoq-iCre mice (Product Code: C001529) using advanced gene editing technologies to express a codon-optimized Cre recombinase (iCre) under the control of the Adipoq gene regulatory elements. Adiponectin, encoded by the ADIPOQ gene, is a crucial protein hormone produced exclusively by fat cells, influencing various pathways related to fat metabolism, such as blood sugar regulation and fatty acid breakdown. The expression pattern of the iCre recombinase is similar to the endogenous gene, and the expression of the endogenous mouse Adipoq gene is not affected.

When Adipoq-iCre mice are crossed with mice containing loxP sites, the progeny mice can undergo Cre recombinase-mediated recombination in their white adipose tissue (WAT) and brown adipose tissue (BAT), enabling targeted studies of these specific fat tissues.

Cre Recombinase Activity in White and Brown Fat

Figure 1: Immunofluorescence (IF) Detection of tdTomato Protein Expression in White and Brown Fat

Adipoq-iCre mice were crossed with Rosa26-LSL-tdTomato mice, which conditionally express the tdTomato fluorescent protein. Immunofluorescence (IF) detection was performed on the white adipose tissue (WAT) and brown adipose tissue (BAT) of the offspring. The results showed a significant presence of tdTomato red fluorescent signals in the adipose tissues of Cre+ mice.

Cre Expression in Other Tissues

Figure 2: Immunofluorescence (IF) Detection of tdTomato Protein Expression in Skeletal Muscle, Skin, Lung, Heart, Testis, and Ovary

The results showed partial red fluorescence in the skeletal muscle, skin, lungs, and bronchi of Cre+ mice. A minimal amount of fluorescence signal was detected in the heart, peri-ovarian fat, ovarian stroma, and corpus luteum of the mice, while no fluorescence signal was observed in the testis.

Key Findings

1. Fluorescent Detection in Adipose Tissues:

Mating Adipoq-iCre Mice with Rosa26-LSL-tdTomato mice results in offspring with significant red fluorescent signals in WAT and BAT. This observed high fluorescence concentration supports effective analysis of fat cell targeting and recombination.

2. Cre Expression in Other Tissues

IF detection revealed partial red fluorescence in skeletal muscle, skin, lungs, and bronchi of Cre+ mice. Minimal fluorescence was observed in the heart, ovarian tissues, and corpus luteum, while no signal was detected in the testes. The observed distribution of fluorescence confirms the model’s specificity for adipose tissue research.

Why Choose Adipoq-iCre Mice?

Adipoq-iCre mice (Product Code: C001529) demonstrate good targeting specificity to adipose tissues, with the expression of Cre recombinase being primarily concentrated in WAT and BAT. Additionally, partial recombination signals are observed in skeletal muscle, skin, lungs, and heart, which, based on histomorphological analysis, are likely adipocytes.  This makes them an ideal choice for targeted research in fat cell biology and related metabolic studies.

Please check our complete list of Cre mouse lines and feel free to contact us for further assistance!

Troubleshooting Fluorescent Detection Issues

Fluorescent detection is crucial for assessing recombination efficiency and gene expression, especially in animal research models. If you encounter undetectable fluorescent signals, consider the following potential causes and solutions:

1. Fluorescence Signal Destruction

(1) Fluorescence Quenching

Problem: During the process of preparing frozen sections, the fluorescence signal is prone to quenching.

Solutions: When observing directly with a fluorescence microscope, ensure to operate in the dark. Alternatively, use immunofluorescence or immunohistochemistry. Additionally, breeding with other fluorescent reporter mice can amplify the fluorescence signal for better observation.

(2) Improper Sample Handling

Improper handling during sample fixation, blocking, or staining steps can lead to weakened or even lost fluorescence signals. Be sure to follow proper handling procedures.

2. Impacts on Gene Expression

(1) Low Recombination Enzyme Expression Levels:

Low expression levels or insufficient activity of the Cre recombinase can result in low recombination efficiency at the target gene flox sites, making it impossible to detect the fluorescence signal. For example, in CMV-Cre mice, the Cre gene is targeted to the X chromosome, and random X-chromosome inactivation can result in the genome being identified as Cre-positive, but the Cre protein not being expressed. Additionally, the CMV promoter is susceptible to epigenetic silencing, which can also prevent recombination from occurring.

(2) Insufficient Induction Conditions:

For example, when tamoxifen is used to induce nuclear expression of Cre, incorrect dosage and timing can affect Cre expression and, consequently, the fluorescence expression of the reporter mice Rosa26-LoxP-Stop-LoxP-tdTomato. Therefore, it is recommended to conduct preliminary experiments to determine the optimal induction conditions before the main experiment, or to try a more efficient Cre expression system.

(3) Impact of Model Design:

For instance, when constructing fluorescent tool mice, the fluorescence gene may be linked downstream via an IRES element. Due to the inherent characteristics of the IRES element, although the genes are co-expressed, the expression level of the gene located after the IRES element is relatively low, thereby affecting fluorescence intensity. If a weak promoter is used to drive expression, detecting the fluorescence signal becomes even more challenging.

Solutions: Ensure adequate Cre recombinase levels and consider pre-experimental optimization for induction conditions. Be aware of potential issues with IRES-linked fluorescence and weak promoters that may affect signal strength.

3. Overlooked Details in Experimental Procedures

(1) Minimize Background Fluorescence Interference

Due to variations in observation position and depth, excitation and emission light can be absorbed by tissues, leading to high background and low signal-to-noise ratio, thereby limiting detection sensitivity. Minimize background fluorescence by using treatments to reduce background noise, such as: performing depilation (hair removal) on the tissue being examined or treating samples with autofluorescence quenchers. Additionally, selecting fluorescent proteins or dyes that avoid the peak wavelengths of autofluorescence can also help. Additionally, setting up control groups can quickly identify the causes of any failures.

(2) Confirm Experimental Materials

Check and verify the quality and suitability of fluorescent antibodies, fluorescent dyes, and other materials.

(3) Confirm Experimental Parameters

Select appropriate wavelengths, such as 554 nm for tdTomato excitation and 581 nm for emission.

(4) Confirm Genotype

It is crucial to use PCR to check the genotype of samples before the experiment. Additionally, use qPCR or Western Blot (depending on the sample) to assess the expression in specific tissues.

By addressing these potential issues, you can maximize the effectiveness of your fluorescent detection and ensure robust results in your research with Adipoq-iCre Mice.

Explore Our Tools for Precision Research

The Adipoq-iCre Mice (Product No. C001529) offer a cutting-edge solution for researchers focusing on metabolic research and adipose cell biology. With their precise targeting of white and brown adipose tissues and integrated fluorescent detection solution, these mice ensure high-quality results and enhanced research capabilities. Overcome common challenges in fluorescent detection and optimize your studies with this specialized model.

For more information on Adipoq-iCre Mice, visit our website today to learn how this innovative model can elevate your research. We’re here to support your research needs and help you achieve accurate and meaningful results. For personalized assistance or to place an order, contact us now and take the next step in your scientific journey!

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