Construction of lentiviral vector containing siRNA sequence of Siglec-1 and verification of inhibition efficiency
10.3760/cma.j.issn.0254-5101.2012.08.013
- VernacularTitle:Siglec-1小干扰慢病毒载体的构建和干扰效率验证
- Author:
Yisong XIONG
;
Chang LI
;
Yi SUN
;
Renqian ZHONG
- Publication Type:Journal Article
- Keywords:
Siglec-1;
Small interfering RNA;
Lentivirus
- From:
Chinese Journal of Microbiology and Immunology
2012;32(8):730-733
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct lentiviral vectors containing small interfering RNA (siRNA) sequence of Siglec-1 and to screen the effective vector.Methods Three fragments of Siglec-1 siRNA were designed and cloned into pGCSIL-GFP lentiviral plasmid.And then the plasmid was cotransfected into 293T cells with pHelper 1.0 and pHelper 2.0 plasmids.Forty-eight hours later,culture supernatant with virus particles was collected and concentrated.Virus titer was determined by 10-fold serial dilution method and virus was transduced into primary cultured mouse bone marrow-derived macrophages (BMM).Flow cytometry and QRT-PCR were used to screen effective vector with inhibition ability.Results Three vshRNA lentiviral plasmids and a control plasmid were constructed successfully and verified by DNA sequencing.Virus titer was between 1×10s TU/ml and 1×109 TU/ml,which was suitable for in vitro and in vivo experiments.The Lv-1 could inhibit Siglec-1 expression effectively in vitro transduction of BMM.Conclusion Lentiviral vectors containing siRNA sequence of Siglec-1 were constructed successfully and an effective vector was screened,which may lay the foundation for using the vector in gene knockdown experiment in vivo.
