Construction and selection of the most efficient siRNA interfering plasmid specific to mouse Qa-1 gene.
- Author:
Zi-He CHENG
1
;
Jian-Pei FANG
;
Hong-Gui XU
;
Qiu-Hui PAN
Author Information
1. Department of Pediatrics, The Second Hospital, Guangzhou Medical College, Guangzhou 510260, Guangdong Province, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Base Sequence;
Cells, Cultured;
Gene Silencing;
Genetic Vectors;
Histocompatibility Antigens Class I;
genetics;
Mice;
NIH 3T3 Cells;
Plasmids;
RNA, Small Interfering;
genetics;
Transfection
- From:
Journal of Experimental Hematology
2008;16(5):1170-1173
- CountryChina
- Language:Chinese
-
Abstract:
This study was purposed to construct three siRNA eukaryotic expression vector specific to mouse Qa-1 gene, to investigate its silencing effect on Qa-1 gene and to select the most efficient siRNA plasmid specific to mouse Qa-1 gene. Three siRNA peptides specific to mouse Qa-1 through siRNA Web design tools of Ambion company were chosed. Jingsai Company helped to complete the siRNA eukaryotic expression vector. The mouse NIH3T3 cells cultured in RPMI 1640 medium with 10% fetal bovine serum were divided into four groups: three groups of the cells were transfected with lipofectamine 2000 reagent and three different siRNA eukaryotic expression vectors, while one group cells were transfected with lipofectamine 2000 reagent alone as negative control. Cells were collected at 24, 48, 72 hours after transfection; the RNA level of Qa-1 was detected by RT-PCR, and the expression position was examined with flow cytometry analysis by using anti-Qa-1 monoclonal antibody. The results indicated that the constructed three siRNA eukaryotic expression vectors were found to be specific to mouse Qa-1 gene. The sequence analysis showed that the sequence was identical to what chosed from web tools. NIH3T3 cells in vitro were adhered in culture that cell shape appeared to change after transfection. RT-PCR and flow cytometry analysis by using anti-Qa-1 monoclonal antibody approved that both Qa-1 RNA and the expression of Qa-1 on cell surface decreased. The decreased levels in the three groups were different. At 24, 48 and 72 hours, the expression of Qa-1 on NIH3T3 cells decreased as in the following: H2-T231: 60.9%, 81.9%, 43.6%; H2-T232: 64.5%, 73.9%, 61.1%; H2-T233: 61.9%, 71.2%, 47.5%. H2-T232 was most efficient one in all three time points. It is concluded that all three siRNA eukaryotic expression vectors selected can successfully suppress the expression of the Qa-1, and from them H2-T232 is most efficient.
