Tamoxifen-¹³C₆

The inducible CreER/CreERT2 system is based on a fusion between Cre recombinase and a modified estrogen receptor ligand-binding domain that retains Cre in the cytoplasm under basal conditions and allows nuclear translocation upon tamoxifen binding, enabling temporal control of site-specific recombination at loxP-flanked genomic loci in vivo or in vitro. Upon tamoxifen administration, CreER translocates to the nucleus and catalyzes recombination between loxP sites, resulting in excision or inversion of floxed DNA segments and enabling temporally defined gene knockout in specific tissues depending on promoter-driven CreER expression. This system has been widely used for inducible gene deletion and lineage tracing in mice, including validation of efficient temporal recombination in developmental and adult tissues using tamoxifen induction paradigms.

Annexin V-based apoptosis assays rely on the detection of phosphatidylserine (PS) externalization from the inner leaflet of the plasma membrane to the outer leaflet, an early biochemical hallmark of apoptosis. Fluorescently labeled Annexin V binds PS in a calcium-dependent manner, enabling identification of early apoptotic cells by flow cytometry or fluorescence microscopy. When combined with a membrane-impermeant DNA-binding dye (e. g. , propidium iodide), this approach allows discrimination between viable (Annexin V−/dye−), early apoptotic (Annexin V+/dye−), and late apoptotic or necrotic (Annexin V+/dye+) cell populations by assessing membrane integrity and PS exposure.

Constitutive germline knockout models are generated by producing a heritable loss-of-function allele in the mouse genome, typically through complete gene disruption in embryonic stem (ES) cells followed by germline transmission or through CRISPR/Cas-mediated editing of zygotes, resulting in offspring that carry a stable null allele in all tissues. Classical approaches rely on homologous recombination in ES cells to introduce targeted gene disruptions, which are then transmitted through chimeric mice to the germline. More recent genome editing strategies use CRISPR/Cas systems to induce double-strand breaks and non-homologous end joining (NHEJ), frequently generating frameshift mutations that abolish gene function, enabling faster generation of knockout alleles directly in embryos. Germline transmission or direct germline editing ensures that the mutation is present in all cells of the resulting animal, allowing systemic functional analysis of gene loss.

Gene editing modify specific sites within the genome through gene deletions, insertions or conversions to study functionally unknown genes or conduct gene therapy. It is also used to change the biological traits of organisms to establish new varieties. Gene editing techniques include zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas 9) (CRISPR/Cas9).

CRISPR-Cas9 knockout in cultured mammalian cells uses an sgRNA to direct Cas9 to a complementary genomic sequence adjacent to a compatible PAM; Cas9 creates a targeted DNA double-strand break, and repair by non-homologous end joining can introduce insertions or deletions that disrupt the coding sequence or functional genomic element. The readout of knockout is detection of edited alleles and loss of gene product or phenotype, commonly by PCR/Sanger-sequence trace decomposition, targeted sequencing, immunoblotting, immunostaining, or flow cytometry when the target protein is detectable at the cell surface.

Apoptosis, also called programmed cell death, is generally characterized by distinct morphological characteristics.

TUNEL staining detects DNA strand breaks by using terminal deoxynucleotidyl transferase to add labeled nucleotides to exposed 3′-OH DNA termini, generating either microscopic staining in fixed cells or tissue sections, or fluorescence/cytometric signal in cell suspensions. TUNEL positivity reflects DNA fragmentation but should not be interpreted alone as definitive apoptosis, because TUNEL can also label necrotic, autolytic, mechanically damaged, or DNA-repair-associated DNA breaks.

Cytotoxicity assays are usually based on the assessment of cell membrane damage, which can also be indirectly detected by measuring cell viability. Detection methods include MTT assay, CKK-8 assay, LDH assay and ATP assay, etc.

CRISPR-Cas9 zebrafish embryo editing introduces targeted double-strand breaks in genomic DNA by delivering Cas9 nuclease with a guide RNA into one-cell-stage embryos; repair by endogenous DNA-repair pathways produces indels or donor-mediated insertions that can be detected by phenotype, PCR-based genotyping, heteroduplex assays, Sanger sequencing, or amplicon sequencing. The readout reflects the frequency and spectrum of edited alleles in mosaic F0 embryos or transmitted F1 animals; because injected embryos can carry multiple alleles, founder screening and sequence confirmation are required before establishing stable mutant lines.

Endocrine diseases often arise from disrupted hormone production, hormone signaling, or target-tissue responsiveness; for diabetes-focused endocrine disease models, insulin signaling regulates glucose uptake, hepatic glucose output, lipid metabolism, and β-cell compensation. Type 2 diabetes develops through interacting defects in insulin resistance, β-cell dysfunction, adipose inflammation, hepatic glucose overproduction, altered incretin signaling, and ectopic lipid metabolism. A major unresolved question is whether endocrine dysfunction is driven primarily by target-tissue insulin resistance, intrinsic β-cell failure, immune/inflammatory stress, or combined multi-organ failure that differs by disease stage.

Autophagy is a process in which eukaryotic cells use lysosomes to degrade their own cytoplasmic proteins and damaged organelles under the regulation of autophagy related gene (Atg). Microtubule-associated proteins light chain 3 (LC3) is recognized as autophagy marker, which transfers from cytoplasmic LC3 (LC3-I) to membrane type (LC3-II). LC3-II/I ratio could be detected by Western Blot and fluorescence microscopy.

Apoptosis is a regulated, generally non-lytic cell-death pathway that removes unwanted, damaged, infected, or abnormal cells through coordinated morphological changes, caspase activation, DNA fragmentation, and membrane remodeling. The intrinsic apoptosis pathway is controlled mainly by mitochondrial outer membrane permeabilization, BCL-2 family proteins, cytochrome c release, apoptosome formation, caspase-9 activation, and downstream executioner caspase-3/7 activation. The extrinsic apoptosis pathway is initiated by death receptors such as Fas, TNFR, and TRAIL receptors, which recruit adaptor proteins and activate caspase-8 before engaging executioner caspases or mitochondrial amplification through BID cleavage. Apoptosis is linked to many phenotypes, including cancer cell killing, tissue homeostasis, immune regulation, neurodegeneration, infection response, and treatment-induced cytotoxicity; unresolved questions include how apoptosis interacts with necroptosis, pyroptosis, ferroptos

Lysosome and acidic-vesicle live-cell staining detects acidic intracellular compartments by using membrane-permeant acidotropic probes that accumulate in low-pH vesicles, including lysosomes, late endosomes, autolysosomes, and acidic phagosomes. LysoTracker staining is commonly used as an intensity-based readout of acidic lysosomal compartment abundance or enlargement, while acridine orange produces green fluorescence in less concentrated compartments and red fluorescence after concentration-dependent accumulation in acidic vesicular organelles. Loss or reduction of acridine-orange red signal can be used as a readout of lysosomal membrane permeabilization or reduced acidic-vesicle integrity. This protocol is designed for live cultured cells and can be adapted for fluorescence microscopy, high-content imaging, plate-reader readout, or flow cytometry when the selected literature supports the readout. Because these dyes report acidotropic accumulation rather than lysosome identity alone,

CRISPR/Cas9 knockout animal modeling uses guide RNA to direct Cas9 to a genomic target, where Cas9 creates a DNA double-strand break; repair by error-prone non-homologous end joining generates insertions or deletions that can disrupt coding sequence and produce knockout alleles. Classic animal-model workflows deliver Cas9 mRNA or Cas9 protein with sgRNA into fertilized zygotes by microinjection or electroporation, then transfer edited embryos into pseudopregnant recipients and genotype founders for target-site mutations.

Macroautophagy is a conserved lysosome-dependent degradation pathway in which cytoplasmic material is sequestered into double-membrane autophagosomes and delivered to lysosomes for degradation and recycling. The pathway supports cellular homeostasis during nutrient limitation, organelle stress, protein-aggregate accumulation, infection, differentiation, and tissue remodeling by coupling cargo sequestration, autophagosome maturation, lysosomal fusion, and degradation of cargo-derived macromolecules. The core molecular sequence includes initiation by nutrient- and stress-regulated autophagy machinery, autophagosome nucleation, LC3/ATG8-family conjugation to autophagosomal membranes, cargo selection through receptors such as SQSTM1/p62, autophagosome-lysosome fusion, and lysosomal degradation. LC3 was identified as a mammalian homolog of yeast Atg8 that localizes to autophagosomal membranes after processing, and p62/SQSTM1 was shown to connect ubiquitinated cargo with autophagic degradati