Stable Cell Line

Abstract

The one problem that is always faced when measuring any construct or device in mammalian cells is the fact that the transfection rate is not always constant. Thus, one never knows how many copies of a construct are actually in the cell. This is why we attempted to create a system that allows a controled integration of our constructs into the genome. To accomplish this we used the FRT/Flp system which is based on homologous recombination by the enzyme flippase at a specific sequence – the FRT site [1]. One of those sequences is on a plasmid that is integrated randomly, but stably into the genome by antibiotic (Zeocin) selection. The other sequence is on the plasmid that contains the construct to be tested. Thus it would be possible to integrate at a specific site on the genome and select cells with the construct via a second antibiotic (Hygromycin). Additionally, the stable integration of an FRT site into the genome allows the characterization of the inserted constructs in the same genomic context thereby providing a normalization of the expression. Lastly, this integration site can also be located by LAM-PCR.

Introduction

Figure 1: Characteristics of FRT site. Cleavage and recombination by FLP takes place within the 8 bp spacer in between repeat 2 and 3. These two repeats are necessary for the reaction. Repeat 1 is present in most of the cases, but not strictly needed for the integration process.

Figure 2: Principle of FRT/Flp recombination. The integration of the GOI is realized by transfecting the cells with both the FRT plasmid containing the GOI and a plasmid which codes for the enzyme flippase. The integration process starting at the 8 bp spacer sequence of the FRT-sites occurs via Flip mediated homologous recombination. The process includes the destruction of the genomic Zeocin resistance and the integration of the Hygromycin resistance originally encoded on the plasmid, thus leading to new selection conditions for stable clones.

Results

We attempted to make a cell line with an FRT site by using the pFRT/lacZeo plasmid (Invitrogen). After transfecting HeLa, U2-OS and MCF-7 cells with the mentioned pFRT/lacZeo plasmid by using the Effectene kit (Qiagen), we selected cells that stably integrated the plasmid by adding Zeocin (100 μg/ml) to the culture medium (DMEM+++). After several days, cells with a Zeocin resistance survive and grow in foci originated from one single cell. Those foci were then picked as described in the cell culture methods. They were expanded into culture flasks and aliquots were frozen. After DNA extraction by use of the High Pure PCR Template Preparation Kit (Roche), LAM-PCR was carried out to localize the integration sites on the genome. Cotransfection of the plasmids pOG44 (encodes for FLP) and p55 (contains CMV promoter and GFP) at a ratio of 9:1 led to the integration of a GFP with a CMV promoter in some of the cells. After this cotransfection the cells were kept under Hygromycin selection (200 µg/ml). There is no promoter in front of the resistance gene for Hygromycin so that cells that randomly and stably integrate the plasmid (as it was done for the pFRT/lacZeo) do not express the resistance. Once again, foci were visible after several days( see Fig. 3).

To properly characterize the integration sites of the pFRT/lacZeo plasmid in the genome we performed LAM-PCR at M. Schmidt's lab (see reference 5 for details) at the nct (national center for tumor diseases) in Heidelberg. Thereby we confirmed that we were able to generate HeLa, MCF-7 and U2-OS cells that stably integrated the FRT-site into their genome. For each cell line there are several bands visible (see Fig. 4). Since the number of bands correlates with the number of unique integration sites [5] there must have been more than one integration of the FRT-vector into the genome of the cells.

Figure 3: One focus of cells that stably integrated a plasmid containing CMV and GFP into their genome. Cells in the focus should all have evolved from a single cell, that successfully passed through homologous recombination at the FRT- site mediated by flippase

Figure 4: Results of second exponential PCR. In all lanes there are several bands visible, indicating that there is more than one FRT-site integrated in each cell line. Negative controls are: 1. untransfected genomic DNA 2. H2O negative control LAM-PCR, 3. H2O negative control first exponential PCR, 4. H2O negative control second exponential PCR. Electrophoresis was carried out on 2% agarose; [http://tools.invitrogen.com/content/sfs/manuals/15628019.pdf 100 bp DNA ladder] was used.

Discussion

Our experiment showed that it is possible to generate cell lines with stable FRT sites. We are aware that our cell lines with multiple FRT sites are not ideal for the characterization of promoters. Nevertheless, we are convinced that a cell line that contains a single integration site can be generated and that it would be very valuable for standardized characterization of BioBrick parts in eukaryotes. Even though those characterizations are more time consuming due to the Hygromycin selection, they are of advantage once established, because the construct cannot be "washed out" as it is observed when dealing with transiently transfected cells. Measurements with our cell lines with multiple integration sites are also possible and favorable over transiently transfected cell lines. For one, it is expected that the number of construct integrations is statistically distributed. Furthermore, one can determine the number of constructs integrated. Since the genomic surroundings of the integration sites can be characterized by LAM-PCR, another (regular) PCR with one primer that is located on the genome and one that is on the other side of the gene of interest (GOI) can be carried out. To generate a cell line with only one integration site, the pFRT/lacZeo should have been linearized before transfection. That way, the transfection efficiency and thus the chance to stably integrate more than one copy would have been lower. Also, linearization of the plasmid is favorable for LAM-PCR, because the sequences directly adjacent to the genomic DNA would be known. Although being very time-consuming, another attempt to create a cell line that stably integrated only one FRT-site would have a great impact for the synthetic biology community.

Detailed Methods

LAM-PCR

Figure 5: Schematic outline of LAM-PCR used to amplify the genomic DNA flanking the FRT-site.The solid phase consists of Streptavidin-coupled magnetic beads which allow for the binding of DNA via biotinylated Primers.

References

[1] Zhou H. Development of site-specific integration system to high-level expression recombinant proteins in CHO cells. Chinese journal of biotechnology 23: 756-62 (2007).

[2] Andrews B. J., Proteau G. A., Beatty L. G. & Sadowski P. D. The FLP Recombinase of the 2μ Circle DNA of Yeast: Interaction with Its Target Sequences. Cell 40: 795-803 (1985).

[3] Senecoff J. F., Bruckner R. C. & Cox M. M. The FLP recombinase of the yeast 2-μ plasmid: Characterization of its recombination site. Proc. Nati. Acad. Sci. USA 82: 7270-7274 (1985).

[4] Holmberg A., Blomstergren A., Nord O., Lukacs M., Lundeberg J. & Uhlén M. The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures. Electrophoresis 26(3): 501-10 (2005).

[5] Schmidt M., Schwarzwaelder K., Bartholomae C., Zaoui K., Ball B.,Pilz I.,Braun S.,Glimm H.,von Kalle C., High-resolution insertion-site analysis by linear amplification–mediated PCR (LAM-PCR). Nature Methods 4(12): 1051-1057 (2005).