ABSTRACT
In scientific research, it is often needed to knock in, knock out, knock down, or overexpress a specific gene in model organisms or specific types of cells to achieve precise regulation of experimental independent variables. In this case, various transgenic mice are required. The cyclization recombinase (Cre) can directly interact with different loxP (locus X over P1) DNA sequences without any cofactors to perform specific gene knock-in or knock-out at specific targets. Because of its advantages of simple action principles, high spatial specificity, and high reorganization efficiency, the Cre-loxP system is widely used in scientific research. Furthermore, the CreERT2 system (mutant of the fusion protein of Cre and estrogen receptor ligand binding domain) and the tetracycline (Tet)-on/off system, derived from the Cre-loxP system, have made the recombination of the target gene occur in temporal-specificity on the basis of spatial-specificity. This dual specificity of time and space is indispensable for research in specific directions such as fear memory and engram cells on the basis of reducing the impacts on experimental animals. Therefore, these derived systems have broad application prospects.
ABSTRACT
The cDNA encoding human Vascular Endothelial Growth Factor 165 (VEGF165) was amplified using RT-PCR from human tonsil tissue and cloned into eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid pcDNA/V was transferred into 293 cells mediated by liposome and the cells stably expressing VEGF were selected under the pressure of G418. ELISA and Western blotting demonstrated that the eukaryotic expression vector pcDNA/V was successfully constructed and its corresponding protein could be expressed efficiently in vitro. Chick Charioallantoic Membrane (CAM) bioassay showed that recombinant protein has biological activity of hVEGF. Model rats with acute myocardial ischemia were used to further study the expression of VEGFin vivo. The model rats were divided randomly into three groups: control group, pcDNA3.1 (+) group and pcDNA/V group. 50microL naked plasmid DNA or saline was intramyocardially injected at three sites into the border zone of infarction. The hearts of rats were excised and fixed histologically, then the infarction sizes were studied by immunohistochemical staining and electron microscope after four weeks. Immunohistochemical staining for VEGF appeared to be negative in control and pcDNA3.1 (+) groups. In pcDNA/V group, myocardial cells in infarction border zone showed positive staining for VEGF in cytoplasm. Ultrastructural anaylsis showed that there were visible hyperplasia of vascular endothilium in pcDNA/V group. The control and pcDNA3.1 (+) groups showed less capillary hyperplasia. In this study, VEGF165 gene was successfully cloned and its protein expressed in vitro and in vivo was of bioactivity, which provides a basis for the further study of biological functions of human VEGF.