Preparation And Detection Of 8S Spider Silk Transgenic Mice

Spider silk protein is a kind of fibrin with a special structure, which is synthesized in the abdomen silk gland of spider. With its light weight, toughness, super contraction, exceptional tensile strength and other high-performance, spider silk protein has a broad application prospects. Spider silk protein octamer is composed of proteins which were translated by the artificial synthesized of spider dragline silk protein gene’s core S sequence. In order to understand the effect of silk protein gene on growing hair properties, we produced transgenic mice by pronuclear microinjection technique. Identifications of the positive transgenic mice were performed by polymerase chain reactions and Southern blot method, and the expressional detections were performed by real-time PCR, Western blot and immunofluorescence.1. Preparation and characterization of 8S spider silk protein transgenic miceSpider silk protein gene can improve the performance of hair to a certain extent. To further explore the effect and the mechanism of the expression of spider silk protein gene, we produced transgenic mice by microinjection that carrying spider silk protein genes specifically expressed in hair follicle cells. Identifications of the positive transgenic mice were performed by polymerase chain reactions and Southern blot analyses. Sixty seven transgenic founder mice were obtained by this method. Three positive mice were identified, the positive rate is 4.47%.In the present study, we successfully prepared 8S spider silk protein transgenic mice, and that is a basic condition to investigate this gene’s functions.2. Expressional detections of 8S spider silk protein transgenic miceAfter getting the mouse models, mRNA level detections were performed at first. The results of real-time PCR indicated 3 positive mice expressed the target gene at the level of mRNA transcription. While there was no expression detected in the control group. The second detections were tested by Western blot in protein level and a-tubulin genes as internal standard. The results indicated 3 positive mice expressed the target gene at the level of translation. Similarly, there was no expression detected in the control group at the protein level. Meanwhile, immunofluorescence assays to observe the hair follicle cells of expressing the target proteins were performed. At last the determinations of transgene copy number were performed by absolute quantitative PCR assays. The results indicated that the copy number of transgenes among the three positive mice were 1.60,1.48 and 2.33 respectively.By the detection means mentioned above, it could be concluded that the target gene has been successfully integrated into mouse genome and stably expressed. The preparation and detection of 8S spider silk protein transgenic mice lays a foundation for the further studies of effects of spider silk protein gene on growing hair properties not only in mice also in other animals.