Figure 1.
Diagram of the streamlined transfection model.
External (extracellular) lipoplexes attach to the surface of the cell, forming clathrin-coated pits, which enter the cell via endocytosis, leading to the formation of endosomes, which either lyse or degrade. This puts the lipoplexes into the cytosol, where they unpack, releasing the mRNA, which translates to unfolded GFP molecules, which then mature (folding and oxidation), to produce active GFP. In addition to the endosomes, the lipoplexes, mRNA, immature and mature GFP are all degraded at set rates.
Table 1.
Rates.
Figure 2.
Diagram of the multiple-lipoplex transfection model.
This includes the same processes as in the streamlined model, except that here the clathrin-coated pits and the endosomes can contain multiple lipoplexes.
Figure 3.
Green dotted (red full) line: deterministic (stochastic) simulation. A) Number of lipoplexes attached to the cell surface. B) Number of lipoplexes contained in endosomes. C) Number of mRNA molecules in the cell. D) Number of GFP molecules in the cell.
Figure 4.
GFP expression: simulation vs. experiment.
A) Computer simulation. B) Experimental time courses.
Figure 5.
Onset time of GFP expression (Simulation vs. Experiment based on time courses shown in Figure 4).
The curves are Gaussian curves based on mean and variance of the full distribution data (shown as a histogram). The dashed green lines show the onset times for simulation with a maturation rate (kM) of 9.23 taken from literature. The solid red lines show the onset times for simulation with a maturation rate (kM) of 5.5. The dotted blue lines show the onset times of the experimental data.
Figure 6.
GFP expression (Simulation vs. Experiment based on time courses shown in Figure 4).
A) Expression Level. Maximum number of GFP molecules with histograms of the distributions and lognormal fits of the histograms as curves. The dashed green lines are from a simulation of the streamlined model. The solid red lines are from a simulation of the multiple-lipoplex model. The dotted blue lines show the experimental data. B) Dose-Response Relationship. Transfection efficiency (TE) is the percentage of cells that exhibited a successful transfection, based on GFP expression. The curve was determined by varying the dosage (µg/ml) in the experiment, and the initial concentration of lipoplexes in the simulation (Lex). The green open triangles are from the simulation of the streamlined model, and the dashed green line is a single-Poissonian fit. The open red circles are from the simulation of the multiple-lipoplex model and the solid red line is a double-Poissonian fit. The solid blue squares are from the experimental data and the dotted blue line is a double-Poissonian fit.
Figure 7.
All plots show a parameter vs. transfection efficiency (TE, red circles) and protein expression (GFP, green triangles). The lines are linear or exponential fits. A) Incubation time. B) Endosome degradation rate. C) Lysis rate. D) Lipoplex size.
Figure 8.
Key aspect of the fully nested transfection model.
In addition to the processes in the multiple-lipoplex model, the fully nested model includes unpacking of lipoplexes and degradation of mRNA within endosomes.