Study On Chimaeric Mice Of Transgenic Spider Dragline Silk Protein Spidroin1 Gene

Spider dragline silk are elastic and tenacious protein fibers with more remarkable mechanical properties than any other fibers, which make them attractive for technical applications. Along with the understanding of the composition, molecular structure and gene sequence of spider dragline silk, reseachers attempt to obtain spider dragline silk protein through transgenic manipulation of goat mammary gland, bacterium, tobacco or potato and bombyx mori, but no satisfied results have been gotten. In this study, PMX-2S-IRES-EGFP retroviral vector was constructed with spider dragline silk protein gene, pIRES2-EGFP vectors and PMX retroviral vector, and 2s proviral stocks were generated by transient transfection of platE cell with vector plasmid. Chimaeric mice were produced by the injection of ES cells, which were infected with proviral stocks, into mouse blastocysts. The research could be used in the study of producing spider dragline silk protein by transgenic animals.The result details were showed as follows:1. Construction of PMX-2S-pIRES2-EGFP retroviral vector plasmidsThe spider dragline silk protein gene 2s was linked with the vector of pIRES2-EGFP, then the 2s-pIRES2-EGFP fragment was linked with retroviral vector PMX. The new retroviral vector PMX-2S-IRES-EGFP includes both dragline silk protein gene and green fluoresent protein gene. After the vector was tested with restriction enzymes, PCR detection and DNA sequencing, the PMX-2S-IRES-EGFP vector were coincident with the theoretical sequences.2. Production of 2s-proviral stocks and determination of viral titersPlatE and 293GP cell were transfected 48h later with PMX-2S-IRES-EGFP retroviral vectors through the cationic liposome method, the quantity of platE cells expressed GFP were higher than that of 293GP cell. Moreover, about 90% platE cells were GFP positive when they were transfected 48h later with PMX-2S-IRES-EGFP retroviral vectors through the calcium phosphate coprecipitation method. The GFP expression and 2S-IRES-EGFP insertion into the genome could be tested in NIH-3T3 cells, which were infected with 2s-proviral stocks, and the viral titers were 2×105cfu·mL-1.3. Passage of mouse embryonic stem cellsThe optimal feeder layer conditions were selected by testing the gain of murine embryonic fibroblasts from different embryonic days, the process time of mitomycin C, the cultural densities, and the cell cultural system of the feeder cells. The gifted ES cell lines kept normal morphology and growth characteristic in our cultural system. ES cell colonies could crowded 60% petri dish after ES cells were freez-thrawed and passaged in 72h.4. Detection of mouse embryonic stem cellGFP-positive ES cells were positive using Alkaline Phosphatase detection and immunofluorescence staining with SSEA1,Oct4,Nanog and Sox2 antibodies.5. ES cells infected with 2s-proviral stocksA large quantity of ES cells were GFP-positive when they were infected 24h later with 2s-proviral stocks. The genomic DNA extracted from GFP-positive ES cells were tested by PCR method, and the expected fragment size (750bp,2S-IRES-EGFP) was indicated. The routine karyotype anaylsis of ES cells showed that about 70% of positive ES cells had the normal number of chromosomes(2n=40)6. Produce mouse chinaere of 2S-positive ES cells2S-positive ES cells were injected into 90 mouse blastocysts which derived from male C57BL/6 strain and female ICR strain, and 77 reconstructed blastocysts were obtained. After 70 of reconstructed blastocysts were transferred into the uterine horns of 5 pseudopregnant female ICR mice,2 mice were pregnancy and yielded 13 chimeric mice,1 lof which were live (7 male and 4 female) and 5 chimeras had ES cell-derived coat color (white plaques).7. Detection of chimerasThe genomic DNA extracted from the mouse tails of 11 chimeras were tested by PCR method, and the expected fragment (750bp,2S-IRES-EGFP) were detected from 6 chimera. The results of southern blot indicated that the genomic insertion (2S-IRES-EGFP) in chimeras were observed, through the hybridization with the labelled GFP probe. The genomic DNA extracted from organs of one positive chimeras were tested by PCR method, and the results indicated that target fragment (250bp) were detected in muscle, heart, liver, lung, stomach, intestine, pancreas and testis.8. Detection of F1 chimerasTransgene-positive male mice detected by PCR and southern blot was mated with female chimeras.15 chimeric mice were yielded,6 of them were males. The genomic DNA extracted from the offspring mouse tails were tested by PCR method, and the result indicated that the target fragment (750bp,2S-IRES-EGFP) were detected from 9 offsprings.In summary, this study establishes a method of producing transgenic spider dragline silk protein gene (2S) chimera mice by the injection of transgenic ES cells, which was infected by PMX-2S-IRES-EGFP retroviral vector constructed with spider dragline silk protein gene, pIRES2-EGFP vectors and PMX retroviral vector. Furthmore, the method could be used in the study of obtaining spider dragline silk protein by transgenic animals.