Visualize and Manipulate Cells with Fluorescent Mouse Tools

ChR2_H134R/EYFP: The "Master Switch" of Optogenetic Neural Circuit Control

ChR2_H134R/EYFP represents a sophisticated fusion of optogenetic control and fluorescent reporting technology. This system combines a light-sensitive ion channel (ChR2) featuring the H134R gain-of-function mutation with enhanced yellow fluorescent protein (EYFP). EYFP emits light at a wavelength of 527 nm, which is well compatible with the blue-light activation of ChR2_H134R, preventing spectral overlap and minimizing signal interference.

Key features:

• Greater photocurrent and increased light sensitivity due to the H134R mutation
• Reduced desensitization compared to conventional ChR2
• Blue light activation (~470 nm) of ChR2_H134R triggers neuronal action potentials
• EYFP marker (527 nm emission) enables clear visualization of cells expressing ChR2_H134R, enabling researchers to confirm the location and distribution of target cells.[1-2]

Figure 1. ChR2_H134R/EYFP Reporter Line Used for the Study of Cardiac Fibroblasts. [3]

GCaMP6f: High-Resolution Visualization of Calcium Transients & Neuronal Activity

Calcium Indicators Are Generally Classified into Two Types: Chemical Calcium Indicators and protein-based Genetically Encoded Calcium Indicators (GECIs). Due to their ability to be specifically delivered to target cell types, GECIs have become the preferred choice for studying brain function. GCaMP6 is a green fluorescent genetically encoded calcium indicator widely used for measuring neuronal activity because of its high sensitivity in detecting neuronal calcium transients.

Molecular mechanism:

• GCaMP6f combines calmodulin (CaM), circularly permutated enhanced green fluorescent protein (cpEGFP), and myosin light chain kinase M13 domain (M13)
• In the absence of calcium ions, cpEGFP remains non-functional
• Calcium binding triggers a conformational change in cpEGFP
• This structural shift enables fluorescent signal emission, signaling neuronal activation
• Capable of detecting single action potential-induced calcium transients under certain conditions ^[4]

Figure 2. GCaMP6f Mice Used for Monitoring Neural Signal Activation in Implantable Neurostimulation Therapy. ^[5]

KikGR and Kaede: "Spatiotemporal Recorders" for Dynamic Cell Fate Mapping

KikGR and Kaede represent a revolutionary advancement in fluorescent reporter technology through their unique photoconvertible properties. Unlike traditional fluorescent proteins, these reporters can irreversibly switch from green to red fluorescence upon exposure to ultraviolet (UV) light. This unique property overcomes the spatial and temporal limitations of traditional fluorescent proteins (e.g., tdTomato), making them powerful tools for cell tracking and fate mapping studies. In mammalian cells, KikGR exhibits higher photoconversion efficiency and is several times brighter than Kaede in both its green and red states.^[6-7]

Advantages over traditional fluorescent reporters:

• Enables precise spatiotemporal tracking of cell fate
• Overcomes limitations of static fluorescent proteins
• KikGR exhibits higher photoconversion efficiency than Kaede
• KikGR demonstrates significantly brighter fluorescence in both green and red states

Figure 3. UV Illumination Alters the Fluorescence Properties of Kaede-Expressing Cells and Kaede Transgenic Mice.[8]

Cyagen offers two specialized mouse models featuring these photoconvertible proteins:

• Rosa26-CAG-KikGR Mouse (Product No.: I001211): Integrates the CAG promoter-Kozak-KikGR-rBG pA expression cassette into the Rosa26 locus, enabling widespread expression of the KikGR protein in vivo.
• Rosa26-CAG-Kaede Mouse (Product No.: I001118): Provides widespread Kaede protein expression in vivo

mNeonGreen: The "Beacon" for Super-Resolution Imaging Applications

mNeonGreen represents the next evolution of green fluorescent proteins, offering dramatically improved performance characteristics for challenging imaging applications.

Enhanced capabilities of mNeonGreen:

• 3-5 times brighter than conventional GFP in live imaging
• Ideal for detecting low-expression tissues and weak expression patterns
• Excitation/emission spectrum of 506/517 nm
• Compatible with multicolor imaging approaches
• Excellent choice for super-resolution microscopy and live-cell imaging
• Effective for tracking endogenous proteins and labeling subcellular structures
• Especially suitable for precise subcellular localization studies [9]

Figure 4. mNeonGreen Protein Exhibits Superior In Vivo Imaging Performance Compared to GFP Protein. ^[10]

The TG-CAG-mito-mNeonGreen Mouse (Product No.: I001183) was generated using transgenic technology to integrate the CAG-mito-mNeonGreen gene expression cassette into the mouse genome. This model is suitable for studying mitochondrial function, localization, and dynamics, making it an ideal tool for investigating subcellular structural dynamics.

"We not only need brighter fluorescence, but also smarter light." From "visualizing cells" to "manipulating life," novel fluorescent proteins are redefining the boundaries of life sciences.

Cre Mouse Lines: Essential Tools for Conditional Expression

The Cre-loxP site-specific recombination system provides researchers with precise control over gene expression in mouse and rat models. Cyagen offers an extensive portfolio of Cre mouse lines – including Cre mice, Cre mice & fluorescent reporter mice, inducible Cre mice, inducible Cre mice & fluorescent reporter mice, Dre mice, and other specialized Cre mouse lines – to support diverse research applications in drug discovery and development.

The Mrc1Cre mouse used in this experiment was provided by Cyagen.

Available Cre mouse categories:

• Standard Cre mice
• Cre mice with fluorescent reporters
• Inducible Cre mice
• Inducible Cre mice with fluorescent reporters
• Dre mice
• Specialized Cre mouse lines

High-Demand Cre Mouse Lines from Cyagen’s Repository

References

[1] Magown P, Shettar B, Zhang Y, Rafuse VF. Direct optical activation of skeletal muscle fibres efficiently controls muscle contraction and attenuates denervation atrophy. Nat Commun. 2015 Oct 13;6:8506.

[2] Ganji E, Chan CS, Ward CW, Killian ML. Optogenetic activation of muscle contraction in vivo. Connect Tissue Res. 2021 Jan;62(1):15-23.

[3] Wang Y, Li Q, Tao B, Angelini M, Ramadoss S, Sun B, Wang P, Krokhaleva Y, Ma F, Gu Y, Espinoza A, Yamauchi K, Pellegrini M, Novitch B, Olcese R, Qu Z, Song Z, Deb A. Fibroblasts in heart scar tissue directly regulate cardiac excitability and arrhythmogenesis. Science. 2023 Sep 29;381(6665):1480-1487.

[4] Park K, Liyanage AC, Koretsky AP, Pan Y, Du C. Optical imaging of stimulation-evoked cortical activity using GCaMP6f and jRGECO1a. Quant Imaging Med Surg. 2021 Mar;11(3):998-1009.

[5] Park DW, Ness JP, Brodnick SK, Esquibel C, Novello J, Atry F, Baek DH, Kim H, Bong J, Swanson KI, Suminski AJ, Otto KJ, Pashaie R, Williams JC, Ma Z. Electrical Neural Stimulation and Simultaneous in Vivo Monitoring with Transparent Graphene Electrode Arrays Implanted in GCaMP6f Mice. ACS Nano. 2018 Jan 23;12(1):148-157.

[6] Ando R, Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12651-6.

[7] Tsutsui H, Karasawa S, Shimizu H, Nukina N, Miyawaki A. Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep. 2005 Mar;6(3):233-8.

[8] Tomura M, Yoshida N, Tanaka J, Karasawa S, Miwa Y, Miyawaki A, Kanagawa O. Monitoring cellular movement in vivo with photoconvertible fluorescence protein "Kaede" transgenic mice. Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10871-6.

[9] Hostettler L, Grundy L, Käser-Pébernard S, Wicky C, Schafer WR, Glauser DA. The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo. G3 (Bethesda). 2017 Feb 9;7(2):607-615.

[10] Shaner NC, Lambert GG, Chammas A, Ni Y, Cranfill PJ, Baird MA, Sell BR, Allen JR, Day RN, Israelsson M, Davidson MW, Wang J. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods. 2013 May;10(5):407-9.