An method for small hairpin RNA expression vector reconstruction for easy single restriction endonuclease identification / 南方医科大学学报
Journal of Southern Medical University
; (12): 1341-1344, 2007.
Article
en Zh
| WPRIM
| ID: wpr-283135
Biblioteca responsable:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To develop an effective method for screening recombinant hairpin RNA expression plasmids using single restriction endonuclease analysis.</p><p><b>METHODS</b>The double-strand DNA fragment containing a ClaI site (the flanking sequences of which were not complementary) was annealed and ligated into small hairpin RNA (shRNA) expression vector pSilencer-4.1 that did not contain ClaI site to construct the circular pSilencer-4.1-ClaI vector. With BamHI and HindIII, the pSilencer-4.1-ClaIwas digested and ligated with the DNA template of green fluorescence protein (GFP) shRNA that did not include a ClaI site. The plasmid DNA of the positive clones was extracted and digested with ClaI, and the inserted DNA sequence of the non-linearized plasmid was identified by sequence analysis.</p><p><b>RESULT AND CONCLUSION</b>DNA sequencing showed that pSilencer-4.1-ClaI was correctly constructed and the plasmids resistant to ClaI digestion were all recombinant vectors encoding GFP shRNA. The constructed pSilencer-4.1-ClaI can be used as a universal vector to construct the shRNA expression plasmid, and the incorporated ClaI sites may allow efficient screening of recombinant shRNA expression vectors.</p>
Texto completo:
1
Base de datos:
WPRIM
Asunto principal:
Plásmidos
/
Factores de Tiempo
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Datos de Secuencia Molecular
/
Secuencia de Bases
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Mapeo Restrictivo
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Ingeniería Genética
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Expresión Génica
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Análisis de Secuencia de ADN
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ARN Interferente Pequeño
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Proteínas Fluorescentes Verdes
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Idioma:
Zh
Revista:
Journal of Southern Medical University
Año:
2007
Tipo del documento:
Article