Transgene expression in stably transgenic organisms is affected by many factors, including the copy number of the transgene in the genome, and by interactions between the transgene and flanking DNA sequences. Transgene copy number has also been shown to effect genetic stability in transgenic plants. Two commonly used methods for transfecting cells are liposome mediated transfection and electroporation. Little is known about the mean transgene copy number or variability of the copy number with these techniques. Quantitative PCR (Q-PCR) has been shown to be an effective method for determining transgene copy number.
The objective of this study was to determine transgene copy number after liposome mediated transfection and electroporation. The mean transgene copy number and variability between individual integration events have been determined.
Q-PCR conditions were optimized for primer annealing temperature and concentration when amplifying a region of the plasmid hEFGFP used for transfection. The quantitative nature of the Q-PCR reaction was confirmed by amplifying 10-fold dilutions of the plasmid and plotting the threshold cycle (CT) value against the log of the plasmid concentration. A correlation coefficient of 1.00 and a calculated PCR efficiency of 93.3% were obtained from this analysis. Caprine fibroblasts were transfected by electroporation or FuGENE® HD reagent with either a circular or linearized hEFGFP plasmid and plated at low density in medium containing Geneticin®. After 10 days of culture, single cell colonies were isolated and expanded. When cultures reached 1-2 million cells, genomic DNA was isolated. Transgene copy number was determined by amplifying genomic DNA from individual clones representing 1x105 cells with Q-PCR. Transgene copy number was calculated by comparing CT values to a standard curve. The transgene copy number for electroporation circular was 2.7 0.75 (n=32) and 1.3 0.65 (n=19) when using a linear DNA construct. FuGENE® HD using a circular plasmid construct generated a gene copy number of 0.5 0.11 (n=14) and 0.64 0.13 (n=16) for the linear plasmid construct. There were significant differences when comparing electroporation circular to all other treatments, however, there were no differences when comparing electroporation linear, FuGENE® HD circular and FuGENE® HD linear to each other.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-04082009-113609 |
| Date | 08 April 2009 |
| Creators | Wilson, Jessica A. |
| Contributors | Dr. Kenneth Bondioli, Dr. Robert Godke, Dr. Gary Hay |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-04082009-113609/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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