Evaluating and Comparing Lipofectomine and Nano Juice Transfection

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Evaluating and Comparing Lipofectomine and Nano Juice Transfection
information of kill curve and DNA extraction ( DNA preparation) using plasmid mini preparation from E-coli culture and using CHO-K1 cells for examination
Abstract
DNA techniques enable scientists to understand the advances in gene functions and gene therapy development. The two transfection methods highlighted in this report are lipofectomine and Nano juice. The protocols of both the techniques are clearly illustrated and the two processes of transfection compared. The efficacy of the two methods depends on the cell type to be transfected, the DNA concentration, among other factors. The objective of the study is to evaluate and compare the efficacy of the two transfection methods, that is, the Nano juice and liposome transfection. Both the methods are non-viral and the cell used is the E. coli. The experiments were recovered from the DNA preparation, cell selection and finally the transfection. In summarizing the results, the Nano juice transfection technique appeared to be superior to the other method. The result in this test was positive while that of the liposome had no transfection. However, there are specific differences that need to be empirically evaluated to enhance the conditions that improve the transfection efficiency and cell survival.
Objective
The experiment was to evaluate and compare the efficacy of the lipofectomine and Nano juice transfection methods.
Introduction
Transfection is the process of introducing nucleic acids into eukaryotic cells by non-viral methods which are liposome transfection, calcium phosphate transfection, and Nano juice/protein or amino based transfection (Karra & Dahm, 2010 p. 6167). The gene transfer technology uses various lipids, chemical or physical techniques as powerful tools to determine how gene functions and expresses itself in the context of a cell. Transfection techniques are applied in various applications including modulation of expression of gene, gene function studies, mutation analysis, biomedical mapping, and making of recombinant proteins (Washbourne & McAllister, 2002, p.568). The three categories of transfection methods are; chemical methods (relying on career molecules), genetically engineered viruses, and physical methods that deliver nucleic acids straight to the cytoplasm (Dalby, et al., 2004, p.99). Transfection methods vary in the types of applicable cells and experiments, and there are wide variety of transfection viability, efficiency, and level of gene expression, among others. The determination of best method relies on factors such as cell type, cellular context, general safety, transgene capacity, desired efficiency, time, and cost.
As a method, transfection obviates or neutralizes the introduction of negatively charged molecules like phosphates backbones of RNA and DNA into cells with negatively charged membranes (Felgner, 2007 p. 741). The cationic lipid-based plasmids and chemicals like calcium phosphate neutralize or create a positive change to the molecules, hence enable easier transfection reagent, especially the lipids, to cross the membrane.
The methods of transfection used in this experiment are the liposome transfection and nanojuice (amino-based transfection). The two are chemical transfection methods and are hereby compared and contrasted depending on the experimental results. The experiments were recovered from the DNA preparation, cell selection and finally the transfection. The figure below gives a summary of the process.
DNA Extraction and Kill Curve
DNA can be extracted from E. coli culture using plasmid DNA extraction. The cell selection is done using the kill curve experiment (the CHO-K1 cells which grows with antibiotics. Optimal selection of stable cell colonies requires a certain concentration of antibiotics. A kill curve shows a dose response due to increasing antibiotics amount required to kill the cells over a time period. Kill curves experiment should be done for each new cell when selecting a new antibiotics or different lots of cell test. The G418 antibiotic was used in this experiment. The G418 exhibits toxicity in eukaryotic cells by hindering ribosomes functions, thereby blocking protein synthesis.

Procedure for Transfection Methods
a. The lipofectamine 2000 procedure
Five sterile Eppendorf’s (in tube labeled A) were made at a concentration of 1ug/ul. All had 4.0 ug. The amount of 250 ul of Opti-MEM 1 were pipetted into the contents of tubes labeled A were gently mixed. Five more sterile Eppendorf’s labelled Brequired concentration of 25 ul of lipofectamine which were split between them. First, 4 ul of lipofectamine was diluted into 248 ul of the Opti-MEM1 medium. Second, 8 ul of lipofectamine was diluted into 246 ul of the Opti-MEM1 medium. Third, 12 ul of lipofectamine was diluted into 244 ul of the Opti-MEM1 medium. Fourth, 20 ul of lipofectamine was diluted into 240 ul of the Opti-MEM1 medium. Fifth, no lipofectamine was placed into 250 ul of Opti-MEM1 medium + CHO-K1 which was the control. Each of the mixtures was incubated for 5 minutes at room temperature. A was placed into B and mixed and left to settle for 20 minutes at room temperature. The 100 ul of the mixture of each tube was then transferred and mixed by the rocking plate motion. All the five mixtures were then incubated for between 18 and 48 hours at 37 degrees Celsius. The concentration of the G418 antibiotics was set to be 400 ug/ml and was added into the solution after 24 hours.
b. The Nano juice procedure
Four dilutions of transfection mixture were prepared into; first, 63 ul Opti-MEM, 1.6 Nano juice booster, 1.6 Nano juice core, and 0.8 plasmid DNA. The second tube had 59 ul Opti-MEM, 3.2ul Nano juice core, 1.6 ul Nano juice booster and 0.8ug plasmid DNA. The third one had 59 ul Opti-MEM, 1.6ul Nano juice core, 3.2ul Nano juice booster and 0.8ug plasmid DNA. The fourth tube had 60 ul Opti-MEM, 3.2ul Nano juice core, 3.2ul Nano juice booster and 0.8ug plasmid DNA. The last test was a negative control that contained cells with MEM. The Opti-MEM and Nano juice were mixed and incubated for 5 minutes at 37 degrees Celsius before adding plasmid DNA. The next incubation was for 15 minutes. The 20 ul of transfection mixture was added to each tube that contained CHO-K cells and the plate gently rocked to distribute the mixture. Finally, the cells were incubated for 72 hours at room temperature at a concentration of G418 antibiotics chosen to be 400ug/ul, which was added after 24 hours.
Antibiotic sensitivity test (Kill curve)
Table 1: Table showing antibiotic sensitivity
AVERAGE
Conc (ug/ml) of G418 % Viability
Day 0 Day 7 Day 14 Day 21 Day 28
0 97 97 81 87 86
100 97 98 95.5 85.5 91
200 97 98 81.5 49.5 73
300 97 94 66 78 83
400 97 96 81.5 67 59
500 97 83 10 0 0
600 97 76 20.5 0 0
700 97 0 0 0 0
800 97 11 0 0 0

Results and Discussions

Figure 2: The kill curve that depicts days 0, 7, 14, 21, and 28

Figure 3: Antibiotics sensitivity test
Antibiotics can generally exist in a medium for transient transfection. As cationic lipids increase permeability of cells, they also reduce the transfection efficiency. Therefore, they are never recommended to be added on the transfection medium. Moreover, avoiding antibiotics in the process also eliminates the need of rinsing cells before transfection. From the curve and bar graph above, higher concentration of the antibiotics is ineffective for the transfection process because more cells die as the number of days increase. It can also be deduced that at 100 ug/ml of G418, the sensitivity of the antibiotics to the cells is considered stable in this case. In the lipofectamine viewed under fluorescence microscope, no transfection was observed.

Figure 4: Lipofectamine Image after 20 hours

Figure 5: Lipofectamine image after 48 hours
The factors that affect the DNA transfection by the use of cationic liposomes are the amount of DNA, incubation time, lipid-DNA complex, and the ratio of DNA and transfection reagent. All these factors should be examined for all experimental cell types to help in optimization and ensuring reproducible results. The optimal DNA varies depending on the nature of the transfected plasmid, number of cells to transect, culture dish size, and the target cell line used. For our case, the results showed that higher DNA levels are inhibitory to the cell type that we used (Maurisse et al., 2010 p.9) and no transfection was observed in the E. coli. Moreover, the amount of the antibiotics used acted as a killer for the cells and no cell expressions were totally observed.
Mini Plasmid DNA quantification and analysis
The plasmid DNA was extracted from the recombinant E. Coli culture. Here, the DNA should be 6.3 kb and 1 kb in the DNA marker.

Figure 6: DNA Analysis
After the mini preparation and DNA purification and putting it on gel, it reaches nearly 5 kb (from 1-7 loading) which was unexpected. Basically, cells need to be transfected at between 40 and 80 % confluence. Too few cells lead to poor cells growth because of lack of cell to cell contact. On the other hand, too many cells result in contact inhibition and may result in resistant to uptake (Kingston, Chen & Rose, 2003, p.8). It is realized that actively dividing cells and presence of enough food, with good incubation leads to better intake of DNA than inactive cells. Also, measuring the DNA concentration using spectrometer, it was found to be 0.041 ug/ml using A260nm. This was too low and would not lead to transfection. This was because the method of liposome transfection is not valid or applicable to all cell types.
The process may involve many risks and the transfected materials may never enter the nucleus in the first place, or may be disrupted before reaching the nucleus. For the cell to prompt this transgene the cells must be compatible and the nucleic acid should be reach the cell nucleus to begin transcription. The concentration of the DNA was too low and this also leads to poor or no transfection. In the Nano juice transfection, the observed results showed a single cell transfection.

Figure 7: Nano juice transfection after 20 hours

Figure 8: Nano juice transfection after 48 hour showing one cell transfection
The test in the experiment that used the Nano juice transfection showed a positive result in one cell after 48 hours. The factors that improve the efficiency in the Nano juice transfection is the concentration of the DNA in the calcium phosphate, the time of incubation, and the duration of plate shock. To get better results in both the tests, DNA optimization must be carried out to test the suitability on the cell to be transfected. Moreover, it is crucial to take note of the incubation time, plate shocking, size of the plate, antibiotics inhibitory levels, and better control.
The recommended transfection method between the two techniques used in this experiment is the protein/amino acid transfection. This is because the method is inexpensive, has high efficiency, is stable and can be used for transient, and is applicable in the wide range of cell types (Yamano, Dai & Moursi, 2010, p.290). Moreover, the method uses components that are easily available and has easy to use protocols. Lastly, the calcium phosphate offers protection against the serum and intercellular nucleases.
Liposome Transfection and Nano juice Transfection
Liposome is a common transfection reagent used in cellular and molecular biology. This is commonly used to enhance the transfection efficiency in plasmid DNA or RNA (mRNA and siRNA) into cell cultures through lipofection. Liposome reagent has lipids which entrap DNA plasmids. On the other hand, Nano juice technique for transfection enables greater efficiency for difficult or hard cell types than the common reagents. This method is less cytotoxic, flexible, compatible with both serum-free and serum-containing media, and is a cutting edge technology (Kingston, Chen, and Rose, 2002 p. 290). The major benefits are; there design to offer maximum transfection, faster research development, simplified protocols, single kit use, and unique formulations which gives results that might not be obtained by other techniques.
Conclusion
The experiment was aimed at using two methods in performing cellular transfection. The liposome and Nano juice transfection methods were used in this experiment. In the results, the lipofactomine transfection showed no cell transfection whereas the Nano juice transfection showed that a positive result in one cell. Optimum results of transfection depends on the variables that include; the cell density, liposome-DNA complex, presence or absence of antibiotics or serum, and DNA concentrations, among others. Antibiotics are not recommended for use during transfection. Moreover, the majority of cells need to be left to incubate for between 24 and 72 to ensure maximum outcome.

References
Dalby, B., Cates, S., Harris, A., Ohki, E.C., Tilkins, M.L., Price, P.J. and Ciccarone, V.C., 2004. Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications. Methods, 33(2), pp.95-103.
Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M. and Danielsen, M., 2007. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proceedings of the National Academy of Sciences, 84(21), pp.741-747.
Karra, D. and Dahm, R., 2010. Transfection techniques for neuronal cells. Journal of Neuroscience, 30(18), pp.6171-6177.
Kingston, R.E., Chen, C.A. and Rose, J.K., 2003. Calcium phosphate transfection. Current protocols in molecular biology, pp.9-1.
Maurisse, R., De Semir, D., Emamekhoo, H., Bedayat, B., Abdolmohammadi, A., Parsi, H. and Gruenert, D.C., 2010. Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC biotechnology, 10(1), p.9.
Tirlapur, U.K. and König, K., 2002. Cell biology: targeted transfection by femtosecond laser. Nature, 418(6895), pp.290-291.
Washbourne, P. and McAllister, A.K., 2002. Techniques for gene transfer into neurons. Current opinion in neurobiology, 12(5), pp.566-573.
Yamano, S., Dai, J. and Moursi, A.M., 2010. Comparison of transfection efficiency of nonviral gene transfer reagents. Molecular biotechnology, 46(3), pp.287-300.