Construction of lentiviral vector containing siRNA sequence of Siglec-1 and verification of inhibition efficiency / 中华微生物学和免疫学杂志
Chinese Journal of Microbiology and Immunology
;
(12): 730-733, 2012.
Artigo
em Chinês
| WPRIM
| ID: wpr-420236
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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Idioma:
Chinês
Revista:
Chinese Journal of Microbiology and Immunology
Ano de publicação:
2012
Tipo de documento:
Artigo
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