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Cao Z.,China Agricultural University | Li Y.,Beijing Gene and Protein Biotechnology Co. | Wen X.,Beijing Gene and Protein Biotechnology Co. | Li Z.,Beijing Gene and Protein Biotechnology Co. | And 4 more authors.
Zygote | Year: 2014

The present study investigated whether a recloning procedure would affect the reproductive performance or the germline transmission capacity of recloned transgenic pigs. This study has also laid the foundation for the development of elite transgenic swine breeds in the future. Recloned transgenic pigs were developed from ear tissue fibroblasts of primary transgenic cloned pigs using a recloning procedure, and their reproductive performance and exogenous gene transmission were analyzed. Two transgenic cell lines with different genetic backgrounds (derived from a female miniature pig and a male Landrace pig) with stable expression of green fluorescent protein (GFP) were established successfully. Furthermore, recloned transgenic embryos were developed to full term successfully. One female Chinese experimental miniature piglet (CEMP) (GFP+) and three male Landrace piglets (GFP+) were delivered naturally. Furthermore, the index values for the reproductive characteristics of the recloned transgenic pigs, such as puberty, gestation period, sperm volume and sperm concentration, were not significantly different from those of conventionally bred pigs. In addition, 53% of the F1 offspring of the recloned transgenic pigs were GFP positive. These results demonstrate that ear tissue fibroblasts from primary transgenic cloned pigs efficiently support the full-term development of recloned transgenic embryos. Furthermore, recloned transgenic pigs maintain normal reproductive performance and stable germline (genetic) transmission capacities. © 2012 Cambridge University Press.

Wang S.,China Agricultural University | Lv X.,China Agricultural University | Zhang K.,China Agricultural University | Zhang K.,University of Florida | And 7 more authors.
Molecular Biology Reports | Year: 2010

PRNP is the gene encoding prion protein whose misfolded and β-sheet-rich isoform is the infectious agent of transmissible spongiform encephalopathy (TSE). TSE, also called prion diseases, cause fatal neurodegenerative and transmissible disorders in human and animals. Among these diseases, bovine spongiform encephalopathy (BSE) has tremendous impact on economy and human health in the world. In the present study, we hypothesize suppression of the PRNP gene expression could raise resistance to BSE in cattle by using vector-based small interfering RNA (siRNA) expression systems. Therefore, the objective was to screen effective DNA-encoding short hairpin RNAs (shRNAs) which could knockdown the PRNP gene expression in bovine fibroblast cells. Human U6 promoter was employed to drive shRNA transcription from the DNA vector, and seven shRNAs, that designed to target coding region and 3′ untranslated region of the PRNP gene, were selected. Four out of seven shRNAs tested were found to be effective in inhibiting the PRNP gene expression, and the most significant suppression level was as much as 62.9% evidenced by real-time RT-PCR. Furthermore, the protein abundance was obviously reduced compared to the control. Overall, the present study demonstrated that vector-based siRNA expression systems is an efficient approach to knockdown the PRNP gene expression in bovine fibroblast cells and thereby provide donor cells for somatic cell nuclear cloning to produce cattle that is resistant to prion related diseases. © 2009 Springer Science+Business Media B.V.

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