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Optimization of Liposome-mediated transfection in sf9 insect cells and analysis of intergration pattern and copy number in stably transformed cell lines

University of British Columbia

With increasing application of DNA transfer technology in human gene therapy and heterologous protein production, various methods of introducing DNA into ex vivo or in vivo cells have been developed in a wide variety of cells. Recently, growing interest has focused on insect cells for production of heterologous proteins. Insect expression systems have been recognized as an easy and rapid system for production of a large quantity of recombinant or therapeutic proteins. Liposome-mediated transfection is an effective non-viral delivery method. However, optimization is essential to achieve maximal production of the heterologous proteins. The goal of this project is to optimize the transgene expression in Sf9 insect cells using lipofection. Transfection variables involved in the efficiency of transgene uptake by cells were examined for optimization. The type of cationic liposomes and the liposome to DNA ratios were the most significant determinants of efficient DNA uptake. The transient expression of the B-galactosidase reporter gene could be improved by 7.8 fold by optimization. Southern blot analyses revealed that the integrated plasmids were predominantly arranged into head-to-tail concatemers in all the stably transformed cell lines except for one. The concatemeric integration was speculated to occur by extrachromosomal homologous recombination between plasmid DNAs and a subsequent illegitimate recombination into the chromosomal DNA by a double-strand break repair pathway. Up to a 27-fold difference in the integrated copy number was observed among 19 stably transformed cell lines by quantitative southern blot analyses. The reporter gene expression in stably transformed cells could be correlated with the integrated copy number in general. In addition to the copy number, the positional effect of the integration loci is likely to play a role in controlling the expression level. A large amount of DNA uptake reflected on the high transient expression is necessary, but not sufficient for high expression in stable cells. However, there appeared to be higher probability of obtaining high-producer cell lines with high transient expression. Therefore, the use of a lipofection protocol, producing high transient expression, is advantageous in producing high producer stable cell lines.

Thesis/Dissertation

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2009-08-13

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http://hdl.handle.net/2429/12185

Zoology

University of British Columbia

UBCV

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