RESUMO
The transgenic mouse strains surfactant protein C-reverse tetracycline transactivator (SP-C-rtTA), club cell secretory protein (CCSP)-rtTA, and tetracycline operator (TetO)-Cre have been invaluable for spatiotemporally regulating gene deletion in the pulmonary epithelium. In this study, we measured the efficiency and specificity of gene deletion that can be achieved in these mice using the Rosa26-eYFP reporter. Triple-transgenic mice (tTg or rtTA/TetO-Cre/Rosa-eYFP) were bred and treated with various doxycycline (dox) regimens to induce gene deletion, which was then quantified in various cell populations by flow cytometry. In these crosses, we found that the TetO-Cre transgene must be transmitted through the female parent to avoid germline gene deletion. With dox exposure during lung development, SP-C-tTg mice deleted in â¼65-75% of alveolar epithelial type II (ATII) cells, but in only â¼45-50% of the integrin ß4+ population, which consisted of club cells and distal lung progenitor cells. In contrast, CCSP-tTg mice deleted in â¼50% of ATII cells and â¼80% of integrin ß4+ cells. Upon dox treatment of adults, deletion in ATII cells and integrin ß4+ cells in SP-C-tTg mice dropped significantly to â¼20% and â¼6%, respectively, whereas CCSP-tTg mice deleted in â¼57% of ATII and â¼40% of integrin ß4+ cells. Interestingly, untreated CCSP-tTg mice also deleted in â¼40% of integrin ß4+ cells, indicating significant leakiness of CCSP-tTg in ß4+ cells. In all mouse groups, minimal deletion occurred in mouse tracheal epithelial cells or in mesenchymal or hematopoietic cells. These data provide the first quantitative, side-by-side comparison of the deletion efficiency for these widely used transgenic mouse strains.
Assuntos
Células Epiteliais Alveolares/metabolismo , Deleção de Genes , Técnicas de Inativação de Genes , Integrases/genética , Pulmão/citologia , Camundongos Transgênicos/genética , Traqueia/citologia , Transgenes , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Doxiciclina/farmacologia , Feminino , Citometria de Fluxo , Genes Reporter , Integrina beta4/análise , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas Luminescentes/genética , Pulmão/embriologia , Masculino , Herança Materna , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Peptídeos/genética , Proteína C Associada a Surfactante Pulmonar , Uteroglobina/genéticaRESUMO
Because genetic manipulation occasionally disrupts the expression of the neighboring genes, the chromosomal locus where the transgene has been integrated should be identified in the use of transgenic organisms. By using a new blend of thermostable DNA polymerase, we established a highly efficient method of inverse polymerase chain reaction for this purpose. By using this protocol, we successfully determined the vector integration sites of 2 mouse lines, NSE-tTA and tetO-Cre, the combination of which is a useful tool in neuroscience research. On the basis of this information, we quantified the relative expression amount of the chromosomal genes adjacent to these transgenes and found that the insertion of the tetO-Cre vector significantly altered the mRNA level of one of the examined genes. Considering the potential risk of the insertion effect, we recommend that the vector integration sites of any transgenic lines should be determined routinely by using this method, and that the expression levels of their neighboring genes should be determined.