[Show abstract][Hide abstract] ABSTRACT: As an attempt to increase the resistance to Newcastle Disease Virus (NDV) and so further reduction of its risk on the poultry industry. This work aimed to build the eukaryotic gene co-expression plasmid of neuraminidase (NA) gene and myxo-virus resistance (Mx) and detect the gene expression in transfected mouse fibroblasts (NIH-3T3) cells, it is most important to investigate the influence of the recombinant plasmid on the chicken embryonic fibroblasts (CEF) cells. cDNA fragment of NA and mutant Mx gene were derived from pcDNA3.0-NA and pcDNA3.0-Mx plasmid via PCR, respectively, then NA and Mx cDNA fragment were inserted into the multiple cloning sites of pVITRO2 to generate the eukaryotic co-expression plasmid pVITRO2-Mx-NA. The recombinant plasmid was confirmed by restriction endonuclease treatment and sequencing, and it was transfected into the mouse fibroblasts (NIH-3T3) cells. The expression of genes in pVITRO2-Mx-NA were measured by RT-PCR and indirect immunofluorescence assay (IFA). The recombinant plasmid was transfected into CEF cells then RT-PCR and the micro-cell inhibition tests were used to test the antiviral activity for NDV. Our results showed that co-expression vector pVITRO2-Mx-NA was constructed successfully; the expression of Mx and NA could be detected in both NIH-3T3 and CEF cells. The recombinant proteins of Mx and NA protect CEF cells from NDV infection until after 72 h of incubation but the individually mutagenic Mx protein or NA protein protects CEF cells from NDV infection till 48 h post-infection, and co-transfection group decreased significantly NDV infection compared with single-gene transfection group (P<0. 05), indicating that Mx-NA jointing contributed to delaying the infection of NDV in single-cell level and the co-transfection of the jointed genes was more powerful than single one due to their synergistic effects.
PLoS ONE 08/2013; 8(8):e71688. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study aimed to clone the peroxisome proliferator-activated receptor γ (PPARγ) gene of the Xuhuai goat and to make transgenic sheep using intratesticular injection, so as to improve the meat quality and flavor by increasing the intramuscular fat content. The coding sequence of the goat PPARγ gene was 1,428 bp, encoding 475 amino acids. Its similarity with other species was 81 (chicken), 89 (mouse), 92 (pig), 98 (cow), and 99% (sheep). The similarity of the corresponding amino acid sequences was 92.9, 97.3, 98.3, 99.6, and 99.8%, respectively. The signal peptide region of the PPARγ protein was not found in this study, demonstrating that the protein is not secreted. RT-PCR and western blot revealed that PPARγ was expressed in vitro, and the protein was localized in the cytoplasm. The PPARγ gene was expressed in F1 transgenic sheep at both the mRNA and the protein levels; the positive ratio was 13.7%.
[Show abstract][Hide abstract] ABSTRACT: Chicken embryonic stem cells (ESCs) were separated from blastoderms at stage-X and cultured in vitro. Alkaline phosphatase activity and stage-specific embryonic antigen-1 staining was conducted to detect ESCs. Then, chicken ESCs were transfected with linearized plasmid pEGFP-N1 in order to produce chimeric chicken. Firstly, the optimal electrotransfection condition was compared; the results showed the highest transfection efficiency was obtained when the field strength and pulse duration was 280 V and 75 μs, respectively. Secondly, the hatchability of shedding methods, drilling a window at the blunt end of egg and drilling a window at the lateral shell of egg was compared, the results showed that the hatchability was the highest for drilling a window at the lateral shell of egg. Thirdly, the hatchability of microinjection (ESCs was microinjected into chick embryo cavity) was compared too, the results showed there were significant difference between the injection group transfected with ESCs and that of other two groups. In addition, five chimeric chickens were obtained in this study and EGFP gene was expressed in some organs, but only two chimeric chicken expressed EGFP gene in the gonad, indicating that the chimeric chicken could be obtained through chick embryo cavity injection by drilling a window at the lateral shell of egg.
[Show abstract][Hide abstract] ABSTRACT: This paper presents cloning of cDNA of lipoprotein lipase (LPL) gene from Xuhuai goat, and the sub-cellular localization analysis through enhanced green fluorescent (EGFP) fusion protein. cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR). Fusion expression vector named pEGFP-LPL was constructed successfully. Then NIH-3T3 cells were transfected with pEGFP-LPL through polyethylene imine and observed under inverted microscope after 48 h transfection. The RT-PCR was performed to analysis the level of expression of mRNA. The complete coding sequence (1,530 bp) of LPL was acquired, and the open reading frame size was 1,437 bp with a capacity to encode 478 amino acids. The prediction of signal peptide region showed that LPL protein contained a short signal peptide with a probability of 100 %, and the signal peptidase cleavage site located between the 23rd and the 24th amino acid with a probability of 65.9 %. RT-PCR results showed the LPL mRNA expressed successfully in vitro. Sub-cellullar localization analysis showed that pEGFP-LPL fusion protein located at the cytoplasm. LPL gene of Xuhuai goat was transfer into sheep by testicular injection. According to detection from different level, the LPL gene was expressed successfully in F(1) generation.