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Yurong Qin,
Hao Chen,
Yani Zhang,
Caiye Zhu,
Bo Gao, Yanhui Yin,
Wei Li,
Qingqing Shi,
Mengmeng Zheng,
Qi Xu,
Jiuzhou Song,
Bichun Li
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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%.
Biochemical Genetics 04/2013; · 0.86 Impact Factor
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ABSTRACT: The objective of this study was to explore the possibility of obtaining stable transgenic animals by intratesticular injection. The recombinant vector pEGFP-H-FABP expressing the goat heart-type fatty acid binding protein and green fluorescent protein was mixed with liposome complexes and randomly injected into the testes of mice. Testicular section, fluorescence, and DNA detection assays of mouse sperm were performed to determine the integration of foreign DNA. The results showed that foreign DNA was successfully expressed in the treated mice. Furthermore, the expression and function of the foreign gene were analyzed in F(1) generation and F(2) generation mice at different levels, with the positive rates of foreign gene transfer into the F(1) and F(2) generations being 4.0 and 30.23 %, respectively. These results strongly support testicular injection as an effective method of producing transgenic animals and indicate that foreign genes can be stably passed on to the offspring. This research has theoretical and practical implications for the improvement in the quality of laboratory animals and for gene therapy.
Molecular Biology Reports 11/2012; · 2.93 Impact Factor
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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.
Molecular Biology Reports 06/2012; 39(8):8439-46. · 2.93 Impact Factor
