Panx1, Panx2, and Panx3 mRNA expression in W3 cells and mouse thymocytes was quantified in triplicate by real-time RT-PCR.
Total RNA from cells being harvested 36 h after transfection or from mouse thymocytes was isolated using TRIZOL reagent (Life Technologies) in accordance with the manufacturer's instructions, and the first-strand cDNA was synthesized with random primers and SuperScript II reverse transcriptase (Life Technologies).
To increase cleavage efficiency, 6 µg of a nuclear extract prepared from mouse WT thymocytes were also added.
No significant abnormalities in T-cell number and population in LNs and spleen were found in Cxcr4 flox/flox/ Lck-Cre mice, although thymocytes were significantly reduced (Additional file 2d), indicating that CXCR4 deficiency does not affect peripheral T-cell development.
However, both GATA3 and Tox were comparably expressed throughout MHCII-specific positive selection, diverging only in the most mature (CD69−CD24loTCRhi) thymocyte subset in 4in8 and wild-type mice, regardless of whether thymocytes were differentiating into cytotoxic- or helper-lineage T cells.
In contrast, migration of Cxcr4 flox/flox/ Lck-Cre mouse-derived thymocytes was greatly reduced.
This was in sharp contrast to wild type mice, in which TUNEL positive thymocytes were rapidly engulfed by CD68+ cells.
By fusion of mouse pluripotent embryonic carcinoma cells with thymocytes, Miller and Ruddle generated hybrids that form carcinomas after transplantation into nude mice, indicating that the differentiated thymocytes were reprogrammed into a pluripotent state.
