Ensuing transfection, Green Fluorescent Protein (GFP) serving as a screenable gene marker took roughly around 48 to 72 hours to reach a detectable threshold of a fluorescent microscope. Several papers have reported that the transfection aid materials including the Lipofectamine 2000 and 3000 reflect a cell-dependent efficacy, which requires further optimization for each cell type (9). Studies demonstrate that the Cos-7 cell line holds the transfection efficiency of roughly 95%; other efforts cite that cells such as Caco-2 display a rather lower efficiency of 75%. Studies focusing on HEK293 cells have elucidated Lipofectamine 3000 results in higher cellular plasmid uptake when compared with other variants such as Lipofectamine 2000 (10). Furthermore, some cell types are intrinsically more resilient to obtaining foreign genetic materials from the environment, neuroblastoma cells and primary astrocyte cells were cited to be among such categories of cells with approximately 10–12% and 5–12% efficiency, respectively (11). Here, we observed that roughly 50 % of cells absorbed the plasmids containing the target and the reporter genes. To facilitate the transfection process we exploited the Lipofectamine 2000. Single cells were observed in 30 wells under a stereomicroscope, proliferated to constitute a clone of 40 cells after 10-12 days (Figure 2).
NOTE: Our results are in concordance with previous studies demonstrating that roughly, a third proportion of the entire wells contains a cell or scattered cells, ensuing the diluting process (7).
NOTE: As cells’ characteristics are distinctive to their type, they may differ in matters of cell-cell interactions; therefore, they may require more time to form. Our experiences unveiled that during the first 5 to 6 days of incubation and frequent media change, the single cell-derived clone begins to form and is observable after 10 to 12 days.
NOTE: To identify the desired genetic modifications the automated Sanger sequencing method is recommended, for high-throughput sequencing analysis, derivative methods of Sanger sequencing including pyrosequencing and illumine-based sequencing have also proven to be highly effective approaches. TA cloning is the method of choice for separating the different alleles of genes to identify the heterogeneity.
NOTE: Sequence analysis without prior selection of single cells and the separation of alleles may lead to witnessing different peaks for the same spot. Interpreting such findings would be an insurmountable task, therefore prior isolations are highly recommended.
Figure 2. Monitoring the process of single-cell colony formation. This illustration presents a clear demonstration of a single (left) and a dual (right) colony formation within a well after 7 days of cultivation. Those containing two colonies should be identified and excluded from further examination.
· Human Embryonic Stem Cells (HEK293 cells): HEK293 cells are one of the most commonly used adherent cell lines. The growing and maintenance of these cells are quite straightforward. The following materials should be provided preceding the commencing of cultivation.
· Dulbecco's Modified Eagle's medium (DMEM): DMEM culture media with either high or low glucose, and with or without L-glutamine. Based on our experience, the variations do not affect the rate of cellular growth.
· Lipofectamine (LFN) 2000 or 3000: LFN compound is known to be safe and commonly applied for gene delivery purposes. Some published papers have argued that the Lipofectamine 3000 transfers the genetic material into the cells more effectively; however, our previous experiences with HEK293/293T and MCF7 demonstrated marginal to no difference between the two compounds. Studies suggest that there may be some difference in cytotoxicity between the LFN 2000 and 3000 when dealing with single-stranded oligonucleotides.
· Opti-MEM medium: This medium contains low serum content that improves the delivery of target genes by reducing the competition between vectors and serum proteins in cell surface binding. Serum-free or reduced media such as Opti-MEM and DMEM devoid of Fetal Bovine Serum (FBS), serve as a diluent of vectors: LFN mixture, and facilitates the cell surface binding and penetration.
· Stereo and fluorescent microscope
· 6, 12, 24, 48 and 96 cell culture plates
· T25 flasks
· 15 ml centrifuge tube
· Micropipette (large blue: 100-1000 μL, small yellow: 10-100 μl, small white: 1-10 μl)
· Multichannel pipet (200-1000 μl)
· 5 or 10 ml Serological pipettes
· Cell counter (Neubauer Chamber or Hemocytometer(
· Water bath (370 C)
· Incubator ( 370 C, 5% CO2)
· Laminar flow hood
Cell culture and transfection procedure:
1) Seed 3.5-4×106 cells per well in a 6-well culture plate containing DMEM devoid of any antibiotics
the day before transfection, which would facilitate reaching 70-85% of confluency prior to
NOTE: Antibiotics similar to serum proteins may interfere with the transfection process. Additionally, antibiotics may lead to an increase in cell death (12).
2) Supplement 250μl Opti-MEM medium with 10μl Lipofectamine and 4μg of the desired vector in
separate 1.5 ml microtubes and further incubated for 20-25 minutes at room temperature.
NOTE: For the majority of cases the appropriate DNA (μg) to Lipofectamine (μl) ratio is 1:2 to 1:3 (according to the manufacture’s instruction). Nevertheless, due to the inherent toxicity of LFNs and distinct cellular characteristics, further optimization of the ratio for every cell line seems to be necessary (13).
3) Gently pipet and mix the vector and Lipofectamine, and further transfer the mixture into plates
4) Replace the Opti-MEM medium with complete growth medium (DMEM, 10% FBS, 1%
penicillin/streptomycin) 4-6 hours following transfection.
NOTE: Cell viability would be compromised if the mixture remains in contact with cells for a longer period. Of note, incubations may slightly vary as a function of the district characteristics of each cell type (14).
Ensuing transfection, screen the cell populations based on the GFP protein expression and Puromycin serving as a selectable marker carried by the vector, identify and separate the cells for further incubation and expansion for 7 days.
1) Trypsinize the cells and transfer them to a sterile 15ml falcon.
2) Pellet the cells via centrifugation at 1500 rpm for 5 minutes.
3) Resuspend the cells in a 1 ml complete growth media.
4) Cellular viability analysis using the Trypan blue method
5) Counting viable cells via Neobar chamber
6) Isolate and resuspended 106 cells in 1 ml growth medium
7) In order to reach a single cell, a serial dilution should be carried out according to the following procedure: 100μl of the original cell suspension should be transferred to a new flask containing 900μl medium, this process dilutes the original sample containing 106 cells by 1:10 ratio, yielding a suspension of 105 populations. Next, 100μl of the flask encompassing 105 cells should be transferred to a subsequent flask and the process should continue accordingly until reaching a population of 103 cells. Next, 100μl of the previous culture should be conveyed to the next flask, however this time, instead of 900μl of media, cells should be supplied with 9 ml of medium, rendering a final concentration of 10 cells per 1 ml medium. Every 100μl derived from the last flask would roughly contain one cell, which should be further transferred into 96-well plates. Figure 1 illustrates how the serial dilution process should be performed.
NOTE: Gentle pipetting throughout the entire process is essential to both preventing the formation of clumped cells and generation of foam. Furthermore, It ensures that cells are homogeneously dispersed in the solution.
NOTE: Proper mixing is a vital step to improve the reproducibility of the serial dilution procedure.
8) Transfer 100μl of the previous step’s result to 96-well plates (Figure 1B).
9) Incubate the cells at 37°C for 6-7 days without any disturbance (Figure 1.C (.
NOTE: After 6-7 days, check the medium for any change in the color, which is a reflection of the pH. In case of acidification and shift in color, substitute half of the medium with fresh and pre-warmed medium to prevent cellular demise. Following reaching desired cellular confluency, cells should be passaged into 48-well plates and as this process further proceeds, the passaging should be carried out into a 24, 12, and 6 well plate in a step-wise manner (Figure 1.D).
Screening and analysis:
1) Extract DNA of each well, amplify DNA by PCR, and ligate PCR product into TOPO cloning vector to separate two alleles of every single cell.
2) Screen and analyze DNA fragments by sequencing.
Figure 3. A comparison between different sequencing results. This illustration is a graphical representation of different sequencing scenarios. A. The chromatogram of a separated allele of an isolated single-cell colony, depicting clean and normal peaks. B. The chromatogram of a separated allele of an isolated single-cell colony, with some noise peaks. C. The chromatogram of a bulk cell population with irregular noise peaks, which may be a result of various mutations in the cell population. Readings of such samples are not valid and demand further refinements in the sample preparation process.