Serum-free transfection of CHO-cells with tailor-made unilamellar vesicles

Abstract

At present, a number of transfection techniques are available to introduce foreign DNA into cells, but still minimal intrusion or interference with normal cell physiology, low toxicity, reproducibility, cost efficiency and successful creation of stable transfectants are highly desirable properties for improved transfection techniques.For all previous transfection experiments done in our labs, using serum-free cultivated host cell lines, an efficiency value of approximately 0.1% for selection of stable cell lines has not been exceeded, consequently we developed and improved a transfection system based on defined liposomes, so-called large unilamellar vesicles, consisting of different lipid compositions to facilitate clone selection and increase the probability for creation of recombinant high-production clones. DNA and DOTAP/DOPE or CHEMS/DOPE interact by electrostatic means forming so-called lipoplexes (Even-Chen and Barenholz 2000) and the lipofection efficiency of those lipoplexes has been determined via confocal microscopy.In addition, the expression of the EGFP was determined by FACS to investigate transient as well as stable transfection and the transfection efficiency of a selection of different commercially available transfection reagents and kits has been compared to our tailor-made liposomes.

Fig. 1

Small-angle scattering pattern of DOTAP/DOPE (A), DOTAP/DOPE + genomic calf thymus DNA 10:1 (B), DOTAP/DOPE + genomic calf thymus DNA 5:1 (C), DOTAP/DOPE + genomic calf thymus DNA 5:1 (65 °C ±2) (D) and DOTAP/DOPE + pBSKII 10:1 (E). Data were recorded at 25 ± 2 °C, if not indicated otherwise. Arrows indicate Bragg peaks corresponding to a Pn3m phase. Circle indicates the remnant lamellar phase

Fig. 2

Small-angle scattering pattern of CHEMS/DOPE (A), CHEMS/DOPE + genomic calf thymus DNA 10:1 (B) and CHEMS/DOPE + pBSKII 10:1 (C). Data were recorded at 25 ± 2 °C

Fig. 3

Intracellular uptake and dissociation of lipoplexes consisting of DOTAP/DOPE and CHEMS/DOPE liposomes labeled with N-Rh-PE (red) and PicoGreen stained pEGFP-N3 (green). Transfected cells were analyzed by confocal microscopy (Leica TCS SP2) after 4 h (A, B) and 72 h (C, D). Panels E, F were shot 72-h post-transfection with an UV-microscope. Lipoplexes composed of DOTAP/DOPE/N-Rh-PE (A, C and E) and CHEMS/DOPE/N-Rh-PE (B, D and F). (A, B) The liposomes were located in the cytoplasm while the DNA moved into the nucleus and was visualized by PicoGreen. Panels C & D show alleviated staining of cells after 72 h due to degradation of the lipid/rhodamine complex. Panel E & F show the EGFP expressing cells after 72 h in UV-excitation in parallel with transmitted light to localize the EGFP expressing cells. PicoGreen, cells with PicoGreen and liposomes or cells served as controls (data not shown)

Fig. 4

Stable transfection experiments along with the tailor-made liposomes. CHO-cells were either transfected with 11 μg DOTAP/DOPE or with 12 μg CHEMS/DOPE. Selection started 24 h after transfection with G418. EGFP expressing cells were quantified at the indicated days post-transfection by detecting the percentage of positive cells (% transfectants) and the relative expression titer indicated by the rfu. (-•-) [%] DOTAP/DOPE transfectants, (-○-) [%] CHEMS/DOPE transfectants, (-▴-) fluorescent DOTAP/DOPE transfected cells and (-▵-) fluorescent CHEMS/DOPE transfected cells

Fig. 5

Comparison of the transient transfection efficiencies of six methods. The CHO-cells were transfected according to the manual and cultivated in 12-well plates. Bars indicate the standard deviation derived from three independent experiments, columns are the positive transfectants [%] and diamonds (-(- -)-) are relative fluorescence