Optimization of PTS2-EGFP Expression in CHO and Vero Cells
Objective: Reporter gene transfer to mammalian cells receives a great deal of attention due to its importance for molecular biology, embryology and developmental biology studies. Among DNA transfer technologies to eukaryotic cells, lipofection is known as the most widely used because of its easy han...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Royan Institute (ACECR), Tehran
2007-01-01
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| Series: | Cell Journal |
| Subjects: | |
| Online Access: | http://celljournal.org/library/upload/article/Ghodratnama.pdf |
| Summary: | Objective: Reporter gene transfer to mammalian cells receives a great deal of attention due to its importance for molecular biology, embryology and developmental biology studies. Among DNA transfer technologies to eukaryotic cells, lipofection is known as the most widely used because of its easy handling procedure, low cell mortality and the natural pathway it undertakes.Materials and Methods: In this study we have examined the transfectability of two cell types: CHO and Vero cells via Lipofection in four different treatments, with combination of exposure duration, 3 and 6 hrs, and different plasmid DNA concentration, 0.5 and 1μgs. A fusion protein expression vector, pUcD2. PTS2-EGFP was used to direct the EGFP protein to peroxisomes after expression of related cDNA. An SPSS analysis was preformed after counting the positive cells.Results: optimum gene expression was found when using 1 μg DNA treated for three hrs for CHO cells, and 1 μg DNA treated for six hrs for Vero cells.Conclusion: The result suggests that CHO lipofection efficiency is significantly increased by both the DNA concentration and exposure time increment; however, an increase in exposure time has less significant effect on low DNA concentration conditions. The same results have been observed for Vero cells. Optimum expression was obtained with highest DNA concentration. |
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| ISSN: | 2228-5806 2228-5814 |