[Construction and identification of Mdr-1-shRNA eukaryotic expression vector].
Zhongguo Shi Yan Xue Ye Xue Za Zhi
; 18(1): 127-31, 2010 Feb.
Article
em Zh
| MEDLINE
| ID: mdl-20137132
ABSTRACT
This study was purposed to construct and identify the short hairpin RNA (shRNA) eukaryotic expression vector for targeting gene mdr-1 which may play an important role in K562/A02. Short hairpin RNA (shRNA) aiming at the target sequence was to synthesized, the 3491-3509, 1539-1557and 3103-3121 nucleotide of mdr-1 mRNA were selected as targets. The selected nucleotides were cloned in the plasmid pGCSilencer-U6-neo-GFP respectively, and the resultant recombinant plasmids were named as pGY1-1, pGY1-2 and pGY1-3. The sequences of the recombinant plasmids were identified by DNA sequencing and PCR electrophoresis. The recombinant plasmids were transfected into the cell line K562/A02 by lipofection. After being transfected for 48 hours, the inhibition of mdr-1 mRNA was detected by real time-PCR, and P-gp expression was detected by Western blot. The results showed that the specific oligonucleotide was cloned into the vector successfully, and the expression of mdr-1 mRNA and P-gp in K562/A02 cells was reduced after transfecting the recombinant plasmid, as compared to the control group. It is concluded that the shRNA eukaryotic expression vector has been successfully established which can inhibit the expression of mdr-1 mRNA, setting up the basis to futher explore the effects of mdr-1 on cell line of K562/A02.
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Base de dados:
MEDLINE
Assunto principal:
Membro 1 da Subfamília B de Cassetes de Ligação de ATP
/
Resistência a Múltiplos Medicamentos
/
Resistencia a Medicamentos Antineoplásicos
/
RNA Interferente Pequeno
/
Vetores Genéticos
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
Zh
Revista:
Zhongguo Shi Yan Xue Ye Xue Za Zhi
Assunto da revista:
HEMATOLOGIA
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
China