Tamoxifen is an estrogen analogue that binds with higher affinity than estrogen to altered estrogen binding domains. There are several ways to administer tamoxifen to mice each with advantages and disadvantages. If precise dosage is not paramount, administration via food is the gentlest to the animal. Keep in mind that induction varies depending on Cre and target genes. Tamoxifen induction regimens should be determined empirically for the specific mouse lines and experimental setup involved. However, the following protocol provides a starting point for the induction of Cre in adult (P56) mice. The following procedure has been used with success to induce robust Cre activity in all major organ systems (validated in ubiquitous Cre /ER expressers, such as B6.
In recent years, the Cre integrase from bacteriophage P1 has become an essential tool for conditional gene activation and/or inactivation in mouse. In an earlier report, we described a fusion protein between Cre and a mutated form of the ligand binding domain of the estrogen receptor (Cre-ER is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages. We demonstrate that a single, intraperitoneal injection of TM into a pregnant mouse at 8.5 days postcoitum leads to detectable recombination in the developing embryo within 6 h of injection and efficient recombination of a reporter gene in derivatives of all three germ layers within 24 h of injection. In addition, by varying the dose of TM injected, the percentage of cells undergoing a recombination event in the embryo can be controlled. Dose-dependent excision induced by TM was also possible in diverse tissues in the adult mouse, including the central nervous system, and in cultured cells derived from the transgenic mouse line. This inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration. 1Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Kyoto, Japan2Department of Nephrology and Blood Purification, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan Background. Podocytes play an important role in maintaining normal glomerular function. A podocyte-specific conditional knockout technology has been established by the use of transgenic mice expressing a podocyte-specific Cre recombinase to clarify the role of genes expressed in the podocytes. However, it may be difficult to examine the role of genes in certain pathologic conditions using conventional podocyte-specific knockout mice because they may be embryonically lethal or exhibit congenital renal abnormality. To introduce a temporal control in the genetic experiments targeting the podocyte, we constructed tamoxifen-inducible Cre recombinase (Cre ER) transgenic mice under the control of podocyte-specific promoter, 2.5-kb fragment of the human podocin (NPHS2) gene. The specificity and efficiency of Cre activity were examined by crossing NPHS2–Cre ER/R26R treated with 4-OHT expressed β-galactosidase specifically in 85% of the podocytes in glomeruli. Expression of Cre recombinase m RNA was mostly restricted to the kidney, especially in glomeruli. In conclusion, we have successfully generated podocyte-specific inducible Cre transgenic mice by tamoxifen administration. These mice allow us to disrupt the genes specifically in the podocytes after birth.
Afimoxifene is a tamoxifen metabolite with both estrogenic and anti-estrogenic effects. Afimoxifene has a higher affinity for the estrogen receptor than tamoxifen, and functions as an antagonist in breast cancer cells. For cre-mediated knockout, tamoxifen tam is a tamoxifen-regulated mercremer fusion protein to activate the formation of the cre recombinases, is used to carry out deletions. Second, also known as creer recombinase, believed to the cre recombinase expression of hemangioblastic mesodermal origin.