Knockdown of the prion gene expression by RNA interference in bovine fibroblast cells.
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.
Wang
Shaohua
S
State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing, 100193, People's Republic of China. shwang510@gmail.com
Lv
Xiaoqing
X
Zhang
Kun
K
Lin
Tonghui
T
Liu
Xiaofang
X
Yuan
Jing
J
Dai
Yunping
Y
Li
Ning
N
D000818:Animals; D002417:Cattle; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D011328:Prions / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism
D011328:Prions; D034741:RNA, Small Interfering; D008156:Luciferases