Identification of Transfection Efficiency Using Qualitative and Quantitative Analyses of Green Fluorescent Protein in CHO Cells
A. Nurul Atikah,
Z.A. Intan Zarina,
M.Y. Nurul Yuziana,
Z.A. Shahrul Hisham
Tranfection is a method that randomly introduce foreign DNA
into cells either to produce genetically modified cells or to produce recombinant
protein. However, efficient transfection is needed for expression of recombinant
protein in mammalian cell. This study aim is to identify the transfection efficiency
of novel expression vectors by qualitative and quantitative analyses. The novel
expression vectors were named, pZAAGFP (without interested element), pZAAH1C
(with integrated element) and pZAAM956 (with enhancer element). Chinese Hamster
Ovary (CHO) cells were transfected with 3 different novel expression vectors
by using Lipofectamine LTX. After 24 h, these cells were observed under confocal
fluorescence microscope. The frequency of both transfected and non-tranfected
cells were determined. Then, the intensity of Green Fluorescent Protein (GFP)
was quantified using fluorescence reader at emission wavelength 506 nm and excitation
wavelength 500 nm. Qualitative analysis showed that all novel expression vectors
were able to express GFP. More than 80% CHO cells had been successfully transfected
with all of the novel expression vectors stated. Quantitative analysis also
showed that GFP intensity of both novel expression vectors named, pZAAH1C and
pZAAM956 were higher than commercial vector and the GFP intensity of pZAAGFP
was about the same as commercial vector. This finding indicates that all novel
expression vectors are able to express the GFP gene and both qualitative and
quantitative analyses can be applied to determine transfection efficiency.
to cite this article:
A. Nurul Atikah, Z. Zulkiflie, M.A.W. Rohaya, S. Sahidan, Z.A. Intan Zarina, M.Y. Nurul Yuziana, M. Zulkifli and Z.A. Shahrul Hisham, 2014. Identification of Transfection Efficiency Using Qualitative and Quantitative Analyses of Green Fluorescent Protein in CHO Cells. Journal of Applied Sciences, 14: 489-492.
August 19, 2013; Accepted: December 18, 2013;
Published: February 10, 2014
Transfection is a method of introducing foreign nucleic acids into cells to
produce genetically modified cells. Transfection is usually used by researchers
to study about the gene function and regulation. Besides that, transfection
also can be used in identifying protein function (Kim and
Eberwine, 2010). Transfection can be classified into transient transfection
and stable transfection. Transient transfection or transient gene expression
in mammalian cells is a method that allows the production of recombinant protein
in a shorter period as opposed to stable transfection which allows production
of recombinant protein in longer period. This is because stable transfection
involves the integration of DNA into the cellular genome. Several approaches
that can be used to transfect expression vectors into the mammalian cells are
biological, chemical and physical approaches (Kim and Eberwine,
2010). Even though many efficient methods are available, modifications at
molecular level are necessary in order to simultaneously increase the production
of recombinant protein and the transfection efficiency by taking the approach
of producing good expression vectors.
Transfection efficiency can be identified by using Green Fluorescent Protein
(GFP) as a reporter gene. GFP was originally discovered back in the early 1960s
when researchers studying the bioluminescent properties of the Aequorea victoria
jellyfish isolated a blue-light-emitting bioluminescent protein called aequorin
together with another protein that was eventually named the green-fluorescent
protein (Shimomura et al., 1962). GFP can act
as fusion protein when transfected into cells. The function and location of
fusion protein was not disturb when they fusion with GFP. Other than that, GFP
also can be used to localize and monitor protein translation when they move
around the cell (Cody et al., 1993). Some protein
need external agents to make them fluoresce. But GFP is able to autocatalytically
form a fluorophore (Chalfie et al., 1994). This
feature has made GFP one of the most widely used proteins as a marker of protein
localization (Tsien, 1998). Aim of this study is to
identify the efficiency of three novel expression vectors that already constructed.
Transfection analysis was analyzed using two analyses (qualitative and quantitative
MATERIALS AND METHODS
DNA preparation: All novel expression vectors named, pZAAGFP (without interested element), pZAAH1C (with integrated element) and pZAAM956 (with enhancer element) were transformed into E. coli TOP10 for plasmid propagation. The plasmids were extracted using QIAprep®Spin Miniprep, (Qiagen, Jerman). Concentration was taken using Biophotometer (Eppendorf, Germany).
Transfection: A day before transfection, cells were seeded at a density of 1.6 x105 cell mL¯1 in alpha minimal essential medium (AMEM) (Biowest, UK) with 5% fetal bovine serum (FBS) (Invitrogen, USA) in 35 mm petri dish. When the cells were 70-80% confluent (usually after 24 h), all the medium was removed and 0.6 mL serum-free medium, Opti-Mem® (Invitrogen, USA) was added into the culture. Two mixtures (A and B) need to be prepared separately for transfection purposes. Mixture A consisted of 2000 ng plasmid dissolved in 100 μL Opti-Mem® and 5 μL Plus Reagent and mixture B was made up of 3 μL Lipofectamine LTX and 100 μL Opti-Mem®. Mixture A was incubated for 15 min at room temperature before being added to mixture B. This cocktail was then incubated again at room temperature for another 15 min, allowing the DNA-reagen PLUS-Reagen Lipofectamine® complexes to form. These complexes were added into the culture. Petri dish was moved back and forward to make sure the complexes were evenly spread. The culture was then incubated for 3 h in CO2 incubator. After 3 h, 1.2 mL of AMEM supplemented with 10% (v/v) FBS were added into the culture. Expression analysis was done after 24 h of transfection.
Qualitative and quantitative analysis: Confocal fluorescence
microscope, LSM 5 PASCAL (Laser Scanning Microscope-Ziess, Germany) was used
to acquire the CHO cells images that had been successfully transfected with
expression vectors where GFP was used in this study as a reporter gene. The
images captured were used to count the number of transfected cells and non-transfected
cells. Then, the percentage of transfected cells was calculated to determine
the efficiency of expression vectors used. The images were taken 24 h following
transfection. Other than imaging analysis, fluorescence reader was also used
to identify the intensity of GFP based on the reading OD. The emission wavelength
used is 506 nm and the excitation wavelength is 500 nm. The reading was expressed
in Relative Fluorescence Unit (RFU).
Statistical analysis: Paired T-test was used to test the difference between the data of each vectors using SPSS version 15.0 (SPSS Inc., USA). Significant value is the data that showed p value less than 0.05 (p<0.05).
RESULTS AND DISCUSSION
The novel expression vectors were named, pZAAGFP (without interested element),
pZAAH1C (with integrated element) and pZAAM956 (with enhancer element). A commercial
vector; phrGFP was used as a positive control. The transfection efficiency of
all these modified expression vectors was compared to commercial vector; phrGFP.
CHO cell was chosen to become a host because CHO cell is a preferred host for
transient transfection (Baldi et al., 2007).
Transfection was then carried out using Lipofectamine reagen which is categorized
under cationic lipids group are positively charged at physiological pH and interact
with the negatively charged DNA through electrostatic interactions. The lipid-DNA
complexes, also called lipoplexes, were internalized through endocytosis and
subsequently released the DNA in the cytoplasm (Dass, 2004).
After 24 h of transfection, images of transfected cells were captured using
confocal fluorescent microscope. Figure 1 showed the images
of transfected CHO cells with all the novel expression vectors. The fluorescence
cells represented the cells that had been successfully transfected and the GFP
gene inside the vectors was expressed. Cells that did not emit any fluorescent
light meanwhile, were non-transfected cells.
||Images of transfected CHO acquired by confocal fluorescence
microscope at magnification 20X after 24 h of transfection. (a) Negative
control, (b) Commercial vector, phrGFP, (c) pZAAGFP, (d) pZAAH1C and (e)
||Qualitative analysis was done by taking pictures using confocal
fluorescent microscope. The percentage of transfected CHO cells (%) was
obtained from the transfection of different expression vectors, phrGFP,
pZAAGFP and pZAAM956. The control was commercial vector, phrGFP. Analysis
was done 24 h after transfection. Statistical analysis using paired T-test
showed there are no significant difference (p>0.05) of all novel expression
vectors to commercial vector, phrGFP
Based on the fluorescence emitted by all of the transfected cultures except
for negative controls culture, this study concluded that all of the novel
expression vectors expressed the GFP gene. This showed that optical imaging
technique is important in imaging optical contrast agents and reporter molecules
(Luker and Luker, 2008).
Numbers of transfected and non-transfected cells were identified and the percentage
of transfected cells was calculated referring to the acquired images. The data
showed that the percentage of transfected cells for all the vectors were more
than 80% (Fig. 2). Percentages of transfected cells using
pZAAH1C was 9% higher while pZAAM956 was 3% higher as compared to commercial
vector. These showed that modified expression vectors efficiency is better than
commercial vector, phrGFP. Paired T-test also showed that there are no significant
different between commercial vector and novel expression vectors (p>0.05).
Other method was done to validate the efficiency of novel expression vectors. Fluorescence reader was used to read the OD at emission wavelength 506 nm and excitation wavelength 500 nm. The cells were seeded in 96-well plate at a density of 1x105 cell mL¯1. The OD reading was taken and represented by Relative Fluorescence Unit (RFU). Figure 3 is the result of fluorescence intensity in RFU for all expression vectors. The graph for fluorescence intensity showed that the fluorescence intensity for all novel expression vectors was higher as compared to commercial vector (Fig. 3). Statistical analysis using paired T-test showed that there are significant differences (p<0.05) between commercial vector, phrGFP and novel expression vector, pZAA M956. Fluoresence intensity for pZAA M956 was 0.6 fold higher than phrGFP. This result indicated that novel expression vector produced better GFP expression as compared to commercial vector, phrGFP.
||Quantitative analysis was done using fluorescence reader.
The OD was read at emission wavelength 506 nm and excitation wavelength
500 nm. The fluorescent intensity was obtained from the transfection of
different expression vectors, pZAAGFP, pZAAH1C and pZAAM956. The control
was commercial vector, phrGFP. Analysis was done 24 h after transfection.
*corresponds to the significant difference (p<0.05) of pZAAM956 to phrGFP
As a conclusion, qualitative and quantitative analyses can be used to identify
the transfection efficiency. This study identified the transfection efficiency
of all novel expression vectors by using these two analyses.
The authors would like to thank the Ministry of Science, Technology and Innovation (MOSTI) for the financial grant (09-05-MGI-GMB002), Ministry of Higher Education (FRGS/1/2011/SG/UKM/02/13 and ERGS/1/2012/SKK11/UKM/02/5) and Universiti Kebangsaan Malaysia (UKM-DLP-2012-025, UKM-DLP-2012-001 and DPP-2013-024) for supporting this research.
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