Tamoxifen (Citrate)

Gene knockout model induced by Tamoxifen Citrate uses Cre-Loxp mice. The Cre recombinase in these mice is driven by a tissue/cell-specific promoter, and the target gene is flanked by Loxp sites. The Cre recombinase is fused with the ligand-binding domain of the estrogen receptor to construct a Cre-ER fusion protein, and the recombination activity of Cre is induced by Tamoxifen Citrate. In the absence of Tamoxifen Citrate, the Cre-ER fusion protein binds to HSP90 and remains in the cytoplasm. After Tamoxifen Citrate induction, the metabolite of Tamoxifen Citrate, 4-OHT, binds to the Cre-ER protein, activates the Cre recombinase, and allows it to enter the nucleus. There, it recognizes the Loxp sites and mediates the knockout of the gene between two (in the same orientation) Loxp sites. (To avoid interference from endogenous estrogens, the ligand-binding domain of the estrogen receptor is modified, transforming Cre-ER into MerCreMer, Cre-ERT, or Cre-ERT2).

Please do not determine experimental conditions based solely on a single reference. It is recommended to consult several relevant studies before conducting the formal experiment and to optimize key parameters through pilot experiments—such as animal strain, age, dosage, frequency and duration of administration, as well as detection time points and indicators.

(1) Dissolution: Tamoxifen Citrate is poorly soluble in water and is generally dissolved in Corn oil (HY-Y1888). It can be heated at 37°C or sonicated to assist dissolution, ensuring complete dissolution. Tamoxifen Citrate should be prepared in the dark and is recommended to be used freshly prepared. Corn oil is relatively viscous. When aspirating the solution, the needle can be left unplugged. After aspirating the solution, attach the needle and expel the air bubbles.
(2) Administration methods: Common methods include intraperitoneal injection, and it can also be administered by oral gavage or subcutaneous injection. When performing multiple intraperitoneal injections, the injection sites should be alternated.
(3) Dosage: The dosage is 50-120 mg/kg, with 3-5 intraperitoneal injections, usually for 5 consecutive days. The dosage for pregnant mice and aged mice (over 6 months old) should be appropriately reduced. Tamoxifen Citrate can affect fetal development and parturition. For pregnant mice, the induction time should preferably be selected in the late pregnancy stage, and progesterone should be injected simultaneously to reduce the probability of miscarriage.
(4) Mice: Mice at 4-6 weeks old may be more sensitive (it is more effective in young mice than in old mice).
(5) Potential toxicity: Prolonged high-level Cre activity can cause potential toxicity. In addition, if an important functional gene is knocked out or using Homozygous Mice, it may also lead to the death of mice. Therefore, close observation should be carried out within one or two weeks after induction. If the mice show abnormalities, the causes need to be analyzed in a timely manner and the experimental plan should be adjusted.
(6) Control: Control group mice should be injected with an equal amount of the corn oil solution of Tamoxifen Citrate (using Tamoxifen Citrate in a genetic background without Cre recombinase) to rule out the influence of Tamoxifen Citrate itself.
(7) Detection window: The time window between Tamoxifen Citrate administration and the start of the experiment should be large enough, at least 7 days, to ensure that the Cre recombinase enters the nucleus and functions.
(8) Separate caging: Animals treated with Tamoxifen Citrate should be caged separately from untreated animals to avoid cross - contamination caused by behaviors such as animals licking the oily Tamoxifen Citrate suspension, grooming their fur, or coprophagy.

Verify the reduced expression/knockout of the target gene at the RNA or protein level through molecular biology and genetic methods (such as Western blot, PCR, and sequencing, etc.).

Tamoxifen Citrate reduces the hexose monophosphate shunt, thereby increasing the incidence of oxidative stress in rat hepatocytes, leading to liver injury.

Molecular Changes: The activities of antioxidant enzymes, including glutathione S-transferase, glutathione peroxidase, and catalase, were significantly ↓, while the content of reduced glutathione also showed a ↓ trend. Concurrently, the levels of thiobarbituric acid reactive substances (TBARS) and liver transaminases, including serum glutamic-pyruvic transaminase (sGPT) and serum glutamic-oxaloacetic transaminase (sGOT), were significantly ↑.