[Gene fusion of egfp & kan and recombinant plasmid construction by red mediated in vivo homologous recombination].
Sheng Wu Gong Cheng Xue Bao
; 23(4): 598-601, 2007 Jul.
Article
em Zh
| MEDLINE
| ID: mdl-17822029
ABSTRACT
Recombineering, a new genetic engineering technology based on high efficiency in vivo homologous recombination, can be used in target DNA knock-in, knock-out and gene cloning. In the process of gene subcloning mediated by Recombineering technique, high-quality target DNA fragments were difficult to obtain using in vitro overlapping PCR,therefore the efficiency of in vivo homologous recombination was severely interrupted. To solve this problem, some technology improvements have been established based on the principle of Red recombinases. The PCR DNA fragments of egfp and kan genes with complementary sequences on the end of each fragment were co-introduced into a pcDNA3.1 vector and Red recombinases containing E. coli DY331 host cells by electroporation. A recombinant plasmid pcDNA3.1-egfp-kan was screened directly by antibiotic marker. The positive rates can reach to 45%. The EGFP gene expression of pcDNA3.1-egfp-kan can be observed by transient transfection of 293 eukaryotic cells.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Recombinação Genética
/
Recombinases
/
Proteínas de Fluorescência Verde
/
Escherichia coli
/
Fusão Gênica
Idioma:
Zh
Revista:
Sheng Wu Gong Cheng Xue Bao
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2007
Tipo de documento:
Article
País de afiliação:
China