[Construction and identification of a multiple myeloma-specific APE1 siRNA expression vector].
OBJECTIVE
To construct a multiple myeloma (MM)-specific APE1siRNA expression vector, and detect the specific knock-down effect of the siRNA on expression of APE1 protein.


METHODS
APE1siRNA cDNA sequence was designed, synthesized and inserted into pSilencer 2.0-U6 linear expression vector. pSilencer APE1siRNA was digested by enzyme EcoRI and BamHI, then linear vector and IgP fragments were conjugated by T4 DNA ligase. pSilencer IgP-APE1siRNA and pSilencer IE-IgP-APE1siRNA were digested by enzyme EcoRI or XhoI. Linear vector and IE or Kappa fragments were conjugated by T4 DNA ligase. Then a MM specific pSilencer K-IE-IgP-APE1siRNA was cloned. The recombinant products were identified by DNA sequencing and enzyme digestions at each step. pSilencer K-IE-IgP-APE1siRNA plasmid was transfected to KM3, HOS, MDA-231 cells by liposome. APE1 gene silence induced by RNAi was analysed by Western blot.


RESULTS
APE1 protein in KM3 cells could be knocked down effectively and specifically by pSilencer K-IE-IgP-APE1siRNA vector. After 2 days, the level of APE1 protein in KM3 cells transfected with siRNA was 0.118 +/- 0.047, while that transfected with plasmid only was 0.988 +/- 0.029. The efficiency of gene silence was 90%.


CONCLUSIONS
A MM specific APE1siRNA expression vector was successfully constructed.
Yang
Zhen-zhou
ZZ
Center of Oncology, Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing 400042, China.
Chen
Xing-hua
XH
Wang
Dong
D
Wang
Ge
G
Xiang
De-bing
DB
D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
D034741:RNA, Small Interfering; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase