ABSTRACT
The G protein-coupled P2Y(2) nucleotide receptor (P2Y(2)R) is upregulated in response to stress and tissue injury and has been postulated to play a role in chronic inflammation seen in atherosclerosis, Alzheimer's disease and Sjogren's syndrome. The role of P2Y(2)R upregulation in vivo is poorly understood, in part due to the lack of a P2Y(2)R overexpressing animal model. The P2Y(2)R overexpressing transgenic rat was generated using a lentiviral vector. Rats overexpressing P2Y(2)R showed a significant increase in P2Y(2)R mRNA levels in all tissues screened as compared to nontransgenic rats. Fura 2 imaging of smooth muscle cells (SMCs) isolated from aorta indicated that the percentage of cells exhibiting increases in the intracellular free calcium concentration in response to P2Y(2)R agonists was significantly greater in freshly isolated SMCs from transgenic rats than wild-type controls. Histopathological examination of tissues revealed that P2Y(2)R overexpressing rats develop lymphocytic infiltration in lacrimal glands and kidneys as early as at 3 months of age. These rats show similarities to patients with Sjogren's syndrome who display lymphocyte-mediated tissue damage. This transgenic rat model of P2Y(2)R overexpression may prove useful for linking P2Y(2)R upregulation with chronic inflammatory diseases, neurodegenerative diseases and Sjogren's syndrome.
Subject(s)
Disease Models, Animal , Lentivirus/genetics , Receptors, Purinergic P2/genetics , Sjogren's Syndrome/genetics , Animals , Animals, Outbred Strains , Aorta/pathology , Aorta/physiology , Female , Gene Expression , Genetic Vectors , Homozygote , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/physiology , Pregnancy , Rats , Rats, Sprague-Dawley , Rats, Transgenic , Receptors, Purinergic P2Y2 , Sjogren's Syndrome/pathology , Sjogren's Syndrome/physiopathology , Transgenes/geneticsABSTRACT
Embryonic stem (ES) cell lines including the C57BL/6 genetic background are central to projects such as the Knock-Out Mouse Project, North American Conditional Mouse Mutagenesis Program, and European Conditional Mouse Mutagenesis Program, which seek to create thousands of mutant mouse strains using ES cells for the production of human disease models in biomedical research. Crucial to the success of these programs is the ability to efficiently cryopreserve these mutant cell lines for storage and transport. Although the ability to successfully cryopreserve mouse ES cells is often assumed to be adequate, the percent post-thaw recovery of viable cells varies greatly among genetic backgrounds and individual cell lines within a genetic background. Therefore, there is a need to improve the efficiency and reduce the variability of current mouse ES cell cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of a C57BL/6 mouse ES cell line by characterizing the membrane permeability characteristics and osmotic tolerance limits. These values were used to predict optimal cooling rates, warming rates, and type of cryoprotectant, which were then verified experimentally. The resulting protocol, generated through this hypothesis-driven approach, resulted in a 2-fold increase in percent post-thaw recovery of membrane-intact ES cells as compared to the standard freezing protocol, as measured by propidium iodide exclusion. Additionally, our fundamental cryobiological approach to improving cryopreservation protocols provides a model system by which additional cryopreservation protocols may be improved in future research for both mouse and human ES cell lines.
