|Public on Mar 31, 2013
|WT ligament tissue
|ligament tissue from wild type mice
|background strain: C57BL/6J
genotype: wild type
|Mesenchymal stem cells (sample 1) derived from mouse embryos were obtained from the Cell Bank RIKEN Bioresource Center, Japan. The cells were suspended in E-MEM and Dulbecco’s Modified Eagle’s medium containing 10% foetal bovine serum (FBS) to a final concentration of 1.5 × 10^5 cells/ml, were seeded into dishes or flasks (2.0 × 10^4 cells/cm2), and were statically cultured at 37°C under 5% CO2. For the expression of murine proteins, the synthesized mouse A sequence was inserted into XhoI of the pCAGGS vector, which was then cloned and transfected into mouse mesenchymal stem cells. Protein expression in the culture supernatant was confirmed by Western blotting (sample 4). The plasmid DNA pCAGGS-XhoI-A-XhoI was used for introduction of the gene into 58 fertilized mouse eggs. These were then implanted into surrogate mouse mothers. A section of tail tissue was cut from 20 of the resulting newborn mice, and DNA was extracted using the DNA mini kit (QIAGEN). PCR was performed to determine whether the samples contained the exogenous gene. Comparison of microarray analyses with mesenchymal stem cells showed homology between the extracellular matrix of the ligament tissues of Wt mice (sample 3), the ligament tissues of mice transgenic for A (sample 2), and mesenchymal stem cell-derived ligament-like tissue.
|Total RNA was prepared from cells using an AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturer’s instructions.
|cRNA was amplified and labelled using a Quick Amp Labelling Kit (Agilent Technologies).
|cRNA was hybridized to a 44K 60-mer oligomicroarray (Whole Mouse Genome Microarray Kit; Agilent Technologies) according to the manufacturer's instructions.
|The hybridized microarray slides were scanned using an Agilent scanner. The relative hybridization intensities and background hybridization values were calculated using Feature Extraction Software version 184.108.40.206 (Agilent Technologies).
|The scanned images were analyzed with Feature Extraction Software 220.127.116.11 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. The raw signal intensities and flags for each probe were calculated from the hybridization intensities and spot information according to the procedures recommended by Agilent Technologies using the Flag criteria in the GeneSpring Software. In addition, the raw signal intensities of two samples were normalized by the quantile algorithm with Bioconductor.
|Jan 23, 2012
|Last update date
|Mar 31, 2013
|New protein from an unexpected ligament