J Korean Endocr Soc.  2006 Oct;21(5):364-369. 10.3803/jkes.2006.21.5.364.

Functional Study of Gene using Inducible Cre System

Affiliations
  • 1Department of Molecular Medicine, Kyungpook National University School of Medicine, Korea.

Abstract

Gene manipulation by disrupting important genes using homologous recombination in mammals has provided important insights into their function and development with regard to disease. However, many questions related to the genetic pathways that regulate cellular differentiation and function remain to be clarified. In particular, analysis of genetic pathways that control embryonic skeletal development is often hindered by the disruption of critical genes that function in early embryogenesis, thereby leading to embryonic or perinatal death and thus preventing study of the role of these genes in skeletal development and physiology postnatally. To overcome this problem gene-targeting methods, using site- and time-specific recombination based methods with the Cre/loxP system, have been used to delete particular genes in specific tissues and stages during development. Thus, the generation and characterization of transgenic mice expressing Cre recombinase, under the control of a tissue-specific and stage-specific promoter, has become prerequisite for study of the physiology and homeostasis of specific tissues during a specific time frame. In this report, we introduce the principles and methods of site-specific and time-specific recombination using the Cre/loxP and inducible Cre system, and discuss the potential applications for applying this system to the study of the development and physiology of the skeletal system.


MeSH Terms

Animals
Embryonic Development
Female
Homeostasis
Homologous Recombination
Mammals
Mice
Mice, Transgenic
Physiology
Pregnancy
Recombinases
Recombination, Genetic
Recombinases

Figure

  • Fig. 1 A, Conditional gene inactiviation by Cre/loxP system. Conditional mice harbor the floxed allele (conditional allele), in which target gene has flanked by two loxP sites. This target gene can be excised and inactivated by Cre recombinase recognizing two loxP sites. B, Transgenic mice expressing Cre recombinase in specific cell types under the control of cell-specific promoter have provided essential tools for studying the function of particular genes in specific tissues. Specific target gene is inactivated by Cre recombinase in specific tissues of the floxed mouse which has normal expression of target gene.

  • Fig. 2 Inducible Cre system. In the absence of appropriate ligand, the Cre recombinase fused estrogen receptor with mutated ligand binding domain is bound to the heat shock protein (Hsp90) and inhibited from the entering the nucleus. Upon administration and binding of estrogen antagonist, tamoxifen (TM) or 4-hydroxytamoxifen (4-OHT) to the mutant estrogen receptor, Hsp90 dissociates from the CreER complex and allows translocation of activated CreER into the nucleus. In nucleus, CreER recognizes the loxP sites in conditional allele of target genes and mediates recombination.

  • Fig. 3 A, Col1a1-CreERT2; Indicible Cre mouse using 2.3-kb collagen promoter. To determine the ability of Cre recombinase to induce recombination specifically in bone of inducible Cre mouse which has osteoblasts- and odontoblasts-specific collagen promoter, inducible Cre mouse was crossed with the ROSA26 mouse with lacZ reporter. Briefly, the LacZ gene, which is inserted in the ubiquitously expressed ROSA locus, is preceded by a transcriptional stop cassette flanked by loxP sites. Thus, in Col1a1-CreERT2;R26R double transgenic mice, LacZ gene should be expressed in bones when Cre recombinase is activated by 4-OHT administration. Cre recombination activated in a ligand-dependent manner occurred in osteoblasts and odontoblasts under the control of collagen promoter. B, Histological analysis of Col1a1-CreERT2;R26R double transgenic mice. Pups containing the Col1a1-CreERT2;R26R double transgene were injected intraperitoneally with TM or 4-OHT and stained with X-gal. They showed the LacZ expression in all bones and the strong X-gal staining in osteoblasts and odontoblast by histology. No X-gal staining was detected in oil-injected control pups.

  • Fig. 4 Breeding strategy for the conditional gene inactivation using inducible Cre mice. Inducible Cre mice are very useful to study the function of genes in specific tissues or at specific stage of development and after birth. The flox/-;CreER mice harboring the tissue-specific CreER transgene and heterozygous for the floxed and null alleles can be generated. Using flox/-;CreER mice, target genes are inactivated in tissues or cells which Cre recombinase expresses after administration of TM or 4-OHT.


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