Preliminary Research Of The Transgenic Chicken Using A Nonviral Vector

Non-viral vectors compared to the viral vectors have some advantages:tolerability、related to their own shortcomings or induction of neutralizing antibodyreactivity and the simplicity of vector construction and identification. It utilized thethree methods of hydrodynamics injection, intramuscular injection and testicularinjection for the research of transgenic chicken with non-viral vectors, which hascertain guiding significance for gene therapy, genetic vaccines and transgenic chickenproduction.1.hydrodynamics injection method: It is10%weight pCAG-EGFP plasmid(40～100μg) that rapidly injected into the vein of6～10day-old cockerel (10～15s).It identified the EGFP expression of heart, liver, spleen, lung and kidney tissues whichwere sampled at8h,1d,3d and5d by fluorescence microscopy observation, PCR, andimmunohistochemistry method. The results showed that it detected the expression ofEGFP in liver, spleen, lung and kidney at8h, but soon disappeared. And the genomicDNA PCR test of heart, liver, spleen, lung and kidney tissues at8h was negative,which showed that EGFP is not integrated into the chicken genome. This methodprovides a powerful tool for the quickly study on gain-and loss-of-function of thechicken liver.2. intramuscular injection method: intramuscular injection40～100μgpCAG-EGFP plasmid injected into the left leg of per6～10day-old cockerel. Itidentified the EGFP expression of heart, liver, spleen, lung and kidney tissues whichwere sampled at8h,1d and3d by fluorescence microscopy observation, PCR, andimmunohistochemistry method. The results showed that it detected the expression ofEGFP in liver and kidney at8h, but soon disappeared. And the genomic DNA PCRtest of liver and kidney tissues at8h was negative, which showed that EGFP was notintegrated into the chicken genome. This method provides a possibility of the quicklyand easily gene therapy.3. testicular injection method: A mixture of liposomes and80μg pCAG-EGFPplasmid was injected into the testis of18weeks healthy rooster with similar weight.48h after transfection, it identified the EGFP expression of the testicular tissue byimmunohistochemical assay. The result is positive, EGFP was successfully expressedin the testis. But EGFP was not detected in the rooster semen which was sampled at30,60and70d by PCR. It may be due to the plasmid loss at spermatogenesis ordegradation of endogenous DNases. Although the reproducibility of testicular injection method is poor, but it still has certain advantages.