Abstract
Genetic tools such as the Cre-Lox reporter system are powerful aids for tissue-specific cell tracking. For example, it would be useful in examining intervertebral disc (IVD) cell populations in normal and diseased states. A Cre recombinase and its recognition site, loxP have been adapted from the bacteriophage for use in genetic manipulation. The reporter mice used here express the red fluorescent protein, tdTomato with flanking LoxP sites (Rosa26 TdTomato mice). We compared two different Collagen type II (Col2) promoter constructs that drive Cre-recombinase expression in mice: (a) Col2-Cre, which allows constitutive Cre-recombinase expression under the control of the Col2 promoter/enhancer and (b) Col2-CreER, which contains a shorter promoter/enhancer region than Col2-Cre, but has human estrogen binding elements that bind tamoxifen, resulting in Cre-recombinase expression. The goal of the study is to characterize Cre-recombinase distribution pattern in Col2-Cre and Col2-CreER mice using tdTomato as reporter in the spine. The expression patterns of these two mice were further compared with Col2 gene expression in the native mouse NP and AF tissues by real-time PCR. We crossed Col2-Cre mice or Col2-CreER mice with the tdTomato reporter mice, and compared the tdTomato expression patterns. Col2-CreER/tdTomato mice were injected with tamoxifen at postnatal day 7 to activate the Cre-recombinase. TdTomato in the constitutively active Col2-Cre mice was detected in the nucleus pulposus (NP), the entire annulus fibrosus (AF), and in cartilaginous endplate and growth plate cells in the lower lumbar and coccygeal spine. In contrast, when Col2-CreER activity was induced by tamoxifen at P7, tdTomato was limited to the inner AF, and was absent from the NP. We have described the differences in Col2 reporter gene expression, in Col2-Cre/tdTomato and Col2-Cre-ER/tdTomato mouse IVD. The information provided here will help to guide future investigations of IVD biology.