Construction and identification of a multiple myeloma-specific APE1 siRNA expression vector / 中华血液学杂志
Chinese Journal of Hematology
; (12): 235-239, 2006.
Article
de Zh
| WPRIM
| ID: wpr-243967
Bibliothèque responsable:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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%.</p><p><b>CONCLUSION</b>A MM specific APE1siRNA expression vector was successfully constructed.</p>
Texte intégral:
1
Indice:
WPRIM
Sujet Principal:
Données de séquences moléculaires
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Séquence nucléotidique
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Transfection
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Clonage moléculaire
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Petit ARN interférent
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Interférence par ARN
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Lignée cellulaire tumorale
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DNA-(apurinic or apyrimidinic site) lyase
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Vecteurs génétiques
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Génétique
Limites du sujet:
Humans
langue:
Zh
Texte intégral:
Chinese Journal of Hematology
Année:
2006
Type:
Article