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.
Artigo
em Chinês
| WPRIM
| ID: wpr-283135
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:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Plasmídeos
/
Fatores de Tempo
/
Dados de Sequência Molecular
/
Sequência de Bases
/
Mapeamento por Restrição
/
Engenharia Genética
/
Expressão Gênica
/
Análise de Sequência de DNA
/
RNA Interferente Pequeno
/
Proteínas de Fluorescência Verde
Tipo de estudo:
Estudo diagnóstico
/
Estudo prognóstico
Idioma:
Chinês
Revista:
Journal of Southern Medical University
Ano de publicação:
2007
Tipo de documento:
Artigo
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