Generation Of VEGF164Overexpression Transgenic Mice By Pronuclear Microinjection And Its Detection

Vascular endothelial growth factor (VEGF) is a dimeric glycoprotein, which is linked by a disulphide bound. VEGF is also a member of the platelet derived growth factors (PDGFs) family. The protein plays an important role in the development of hair follicles and the growth of hair fibers. To further investigate the gene’s function, in this study we produced VEGF164transgenic mice by pronuclear microinjection method, and the positive transgenic mice were screened by polymerase chain reaction (PCR) assay. The relative expression quantities of VEGF mRNA were tested by real-time PCR, while the protein expression levels of VEGF were performed by western blot. Morphology and quantities of murine hair follicles were observed in paraffin section.1. Transgenic mice were produced by pronuclear microinjectionAs an important experimental animal, transgenic mouse could be used to study gene regulation, ontogenesis and disease. For the preparation of transgenic mice, pronuclear microinjection is widely used and very successful. This method includes preparation of exogenous gene, obtaining of pronucleus stage embryos, pronuclear microinjection, and embryo transfer. In this research, we obtained59K14/VEGF transgenic mice and87Kap6-1/VEGF transgenic mice by using pronuclear microinjection, and from each group we identified5positive transgenic mice by PCR, with the positive rates of8.5%and5.8%, respectively. The exogenous genes in the original generations of transgenic mice were all inheritable stably. The VEGF164transgenic mouse model was successfully established.2. Identification of K14/VEGF and Kap6-1/VEGF transgenic micePositive transgenic mice and littermate controls were identified by real-time PCR, western blot, and paraffin methods, etc. Compared to the wild type littermates, the maximum expression value of VEGF mRNA is6.25folds and6.67folds higher in the K14/VEGF and KAP6-1/VEGF transgenic mice, respectively, and the protein expression levels increased1.68folds (K14/VEGF) and1.64folds (KAP6-1/VEGF). Both the hair diameter and the follicle number of transgenic mice increased dramatically from paraffin section observation. This study revealed that VEGF could promote the hair follicles’growth.