Generation Of Transgenic Wuzhishan Minipigs Expressing Monomeric Red Fluorescent Protein By Somatic Cell Nuclear Transfer

Red fluorescent protein and its variants enable the expression, localization and interaction dynamics of proteins to be visualized in vitro in real-time. Fluorophores with higher wavelengths are preferred since they efficiently penetrate tissues and have less toxic emissions. A newly generated fluorescent protein marker, monomeric red fluorescent protein (mRFP1), is particularly useful because of its rapid maturation and minimal interference with GFP and GFP-derived markers. To our knowledge for the first time, we generated the pCX-mRFP1-pgk-neoR construct to evaluate the ability of mRFP1to function as a fluorescent marker in transgenic Wuzhishan minipigs.Wuzhishan minipigs have small body type, early sexual maturity, and the same type O blood with the persons. The tissue metabolic and biochemistry synthesis reflect for medicine in the liver and kidney are similar to humans, as an experimental animal model has been widely studied. Production excellent characteristics and disease resistance of Wuzhishan miniature pig by somatic cell nuclear transfer (SCNT) is a fast and effective method, therefore exogenous gene into nuclear cell is very important.This study was designed to examine the biological characteristics of non-transfected and transfected ear fibroblasts with pCX-mRFPl-pgk-neoR which involved the observations of cell morphology and chromosome nuclear type analysis, compared the transfection efficacy of several transfection reagent, methods, doses of every transfection reagent, exposuring time of the WMP of the DNA and transfection reagent, screening concentration of G418. After we investigated the exogenous DNA had been integrated into genome by PCR, the transgenic embryos were generated by SCNT of nuclei isolated from ear fibroblasts expressing mRFPl, compared the ranging cleavage and blastocyst of cloned embryos reconstructed with transfected or non-transfected donor cells. Finally generate transgenic Wuzhishan minipigs expressing of monomeric red fluorescent protein by SCNT.Conclusion:Our optimized transfection conditions as well as cells were60%-80%confluent, used an electroporation-based Lonza Nucleofector system, the rate of transfection was83.33%, the surviving cells exhibited transgene expression48h after nucleofection when program U-023was used. This efficiency was higher than that of transfection of WMPs with FuGENE(?) HD (ca.2-65%) or LipofectamineTM2000(ca.2-9%). Screening concentration of G418was200ug/ml. The analyses of non-transfected and transfected WMP normal chromosome was92%and80.5%(46/50and41/50, respectively), have no significant difference. Donor cells transfected with pCX-mRFPl-pgk-neoR gene were used in nuclear transefer, the overall developmental rates of transgenic SCNT embryos showed the normal cleavage rate was83.08%, blastocyst formation rate was11.65%. Our results imply that ubiquitous expression of mRFPl at Wuzhishan minipigs ear fibroblast cells does not affect the development of reconstructed embryos by SCNT. A total of685transgenic embryos were transferred to three recipients, two of which became pregnant. Of these, one recipient had six aborted fetuses, and the other gave birth to four offspring. All offspring expressed the pCX-mRFP1-pgk-neoR gene as shown by PCR, fluorescence in situ hybridization analysis and southern blotting. The transgenic pigs expressed mRFPl in all organs and tissues at high levels. These results demonstrate that Wuzhishan minipigs that efficiently express mRFPl can be generated, meanwhile provides information for establish the system to get transgenic minipigs by SCNT. Monomeric red fluorescent protein in transgenic Wuzhishan minipigs could provide a useful model for donors long-term tracking of cells and tissues in transplantation experiments. The transgenic animals as excellent models may have applications in medicine and agriculture.