Packaging and concentrating of high titer of retrovirus containing IL-4RA.
- Author:
Gui-Lan WANG
1
;
Xiang-Teng LIU
;
Ji-Rong LU
Author Information
- Publication Type:Journal Article
- MeSH: Asthma; therapy; Genetic Therapy; Genetic Vectors; genetics; Humans; Polymerase Chain Reaction; Receptors, Interleukin-4; antagonists & inhibitors; Retroviridae; genetics; Transfection; Virus Assembly
- From: Chinese Journal of Contemporary Pediatrics 2009;11(9):761-764
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a recombination retroviral expression vector pLNC-IL-4RA with high efficiency transfection and carrying a screening label.
METHODSIL-4RA was inserted into retroviral vector pLNC-Laz to get recombination retroviral expression vector pLNC-IL-4RA and then transfected into packaging cell line PA317 by liposome transfection. The transfected PA317 cells were obtained and amplified by G418 pressure screening. The cell culture supernatants containing viruses were harvested and the viral titer was determined by NIH3T3 cells infection.
RESULTSThe G418 resistant clones were titrated and checked for the presence of replication virus. The results showed that the highest titer of viral supernatant was 1 x 10(4) CFU/mL. Genome DNA isolated from the cell clone of the highest titer showed the function gene, IL-4RA cDNA, had integrated into the genome of host cells verified by PCR.
CONCLUSIONSThe recombination retroviral vector pLNC-IL-4RA encoding IL-4RA after packaging PA317 cells have higher viral titer. This provides a basis for gene treatment of asthma.
