Mol Biotechnol. 2015 Dec;57(11-12):1018-29.
End Joining-Mediated Gene Expression in Mammalian Cells Using PCR-Amplified DNA Constructs that Contain Terminator in Front of Promoter.
- 1Innovation Center, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan. email@example.com.
- 2Yamaguchi University Biomedical Engineering Center (YUBEC), Tokiwadai, Ube, 755-8611, Japan. firstname.lastname@example.org.
- 3Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan.
- 4Innovation Center, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan.
- 5Department of Environmental and Preventive Medicine, Faculty of Medicine, Oita University, Yufu, 879-5503, Japan.
- 6Department of Applied Chemistry, Faculty of Engineering, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan.
- 7Yamaguchi University Biomedical Engineering Center (YUBEC), Tokiwadai, Ube, 755-8611, Japan.
- 8Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube, 755-8611, Japan. email@example.com.
- 9Yamaguchi University Biomedical Engineering Center (YUBEC), Tokiwadai, Ube, 755-8611, Japan. firstname.lastname@example.org.
Mammalian gene expression constructs are generally prepared in a plasmid vector, in which a promoter and terminator are located upstream and downstream of a protein-coding sequence, respectively. In this study, we found that front terminator constructs-DNA constructs containing a terminator upstream of a promoter rather than downstream of a coding region-could sufficiently express proteins as a result of end joining of the introduced DNA fragment. By taking advantage of front terminator constructs, FLAG substitutions, and deletions were generated using mutagenesis primers to identify amino acids specifically recognized by commercial FLAG antibodies. A minimal epitope sequence for polyclonal FLAG antibody recognition was also identified. In addition, we analyzed the sequence of a C-terminal Ser-Lys-Leu peroxisome localization signal, and identified the key residues necessary for peroxisome targeting. Moreover, front terminator constructs of hepatitis B surface antigen were used for deletion analysis, leading to the identification of regions required for the particle formation. Collectively, these results indicate that front terminator constructs allow for easy manipulations of C-terminal protein-coding sequences, and suggest that direct gene expression with PCR-amplified DNA is useful for high-throughput protein analysis in mammalian cells.
KEYWORDS: FLAG-tag; Gene expression; Mutation; Non-homologous end joining (NHEJ); Peroxisome localization signal sequence; Polymerase chain reaction (PCR); Transcriptional terminator; Transfection
Transfection of PCR-amplified DNA in mammalian cells
Gene amplification by PCR is an easy and quick method compared to the E. coli plasmid preparation. However, PCR-amplified DNA is not commonly used for gene expression experiments in mammalian cells. There may be unknown problems in the transfection experiments using PCR-amplified DNA in mammalian cells. To compare the gene expression levels between plasmid and PCR-amplified DNA, pCMV-GLuc (luciferase, New England Biolabs) plasmid and the PCR-amplified DNA encompassing CMV promoter to a terminator derived from pCMV-GLuc were used for transfection of COS7 cells under the same protocol, and the luciferase activities were measured (Figure 1). The gene expression from PCR-amplified DNA was remarkably lower than that from plasmid. Therefore, the lower expression is one of the problems when PCR-amplified DNA is used for gene expression experiments in mammalian cells.
In addition, several drawbacks are anticipated in the application of PCR-amplified DNA for mammalian cell transfection. One might be the difficulty of large amount of DNA preparation by PCR. Often, a few µL of 0.5-1 mg/µL plasmid DNA per well are used in mammalian transfection experiments in 24-well or 6-well plates. However, DNA concentrations prepared by PCR are about 20-50 ng/mL in 10-50 mL of reaction mixtures. Unless PCR-amplified DNA is concentrated, 0.5-1 µg/µL of DNA cannot be obtained. If PCR-amplified DNA is attempted to use directly for transfection experiments without concentration process, 96-well plate culture is fitting.
Another drawback is the construction of gene constructs. If researchers want to express a gene of interest, it should be inserted between a promoter and a terminator. In E. coli plasmid cloning, the genes are inserted at the multi-cloning site between a promoter and a terminator in plasmid vector. In PCR method, fusion PCR may be used for the construction. If a terminator could be attached to the gene of interest only by PCR using an oligonucleotide primer, the gene construction process becomes easier. We introduce here that an efficient transfection method of PCR-amplified DNA to mammalian cells in 96-well plates and novel gene construction methods by PCR.
Figure 1. Gene expression from plasmid and PCR-amplified DNA. COS7 cells were transfected using plasmid DNA (pCMV-GLuc) and PCR-amplified DNA fragment (CMV-GLuc). At 24, 48, 72 and 96 h after transfection, secreted Gaussia luciferase activity was measured. RLU was indicated counts/mL of culture fluid/s.
A 96-well transfection method using a small amount of DNA
We improved chemical transfection of mammalian cells in 96-well plate culture. Two well-known chemicals, polyethylene glycol (PEG) and RNA, were found as reagents that enhance commercial chemical transfections (1). The enhancer activity of PEG was found by chance. We have been examining chemical transfection methods using plasmid DNAs. When we tested linearized plasmid DNA for a transfection experiment, a restriction-enzyme-digested plasmid DNA and a re-circularized plasmid DNA after ligation reaction as a control were used. Mammalian cells transfected with the re-circularized plasmid expressed a reporter gene higher than the original circular plasmid. We noticed that one of the components in the ligation reaction buffer, PEG, was the cause of enhancing effect.
The idea of RNA was obtained from yeast transformation. In yeast transformation, PEG and lithium acetate are used with carrier DNA or RNA as an enhancer material (2). Therefore, we attempted to mix RNA for mammalian chemical transfection and obtained a result that RNA, especially transfer RNA, played an enhancer role. Luckily, PEG and tRNA synergistically enhanced mammalian transfection (Figure 2). These studies of the details were described in reference 1.
Figure 2. Synergistic enhancer effect of PEG and tRNA on mammalian cell transfection. Plasmid DNA (pCMV-GLuc-SVter) was transfected into HEK293 cells with or without polyethylene glycol (PEG3350: polyethylene glycol MW 3350) and/or yeast transfer RNA (tRNA) by the method described. At 24 h after transfection, secreted Gaussia luciferase activities were measured. RLU was indicated counts/mL of culture fluid/s.
A functional terminator is essential for the efficient expression of linear DNA but not for plasmid DNA
The difference between circular plasmid DNA and PCR-amplified linear DNA is the presence or the absence of DNA ends. Since circular DNA has no end, mRNA transcription from a promoter will last to the site of a terminator somewhere in a plasmid. However, linear DNA has the end of mRNA transcription even without a terminator. We analyzed the importance of terminator in linear DNA constructs. We found that no expression was observed when PCR-amplified DNA was used without terminator. In the case of circular plasmid, the functional terminator is not necessary at close downstream from a coding sequence (Figure 2 in reference 1). Therefore, the importance of terminator cannot be evaluated when circular plasmid was used as reporter because other terminators or terminator-like sequences might be functional even those were located far from the coding sequence. We characterized terminator sequences of SV40 and rabbit b-globin gene in a linear DNA expression system and found that 60-100 bp segments were sufficient for the expression. Because these sizes are short enough for synthetic oligonucleotide primer design, the terminator sequences were attached by PCR using relatively long primers containing the terminator sequences at the 5’ side of annealing sequences. We named these oligonucleotides ‘the terminator primers’, which allowed easy construction of recombinant genes. We described for details in reference 1.
Setting terminator in front of promoter in linear DNA constructs.
Transcription starts from promoter and ends at terminator. This is common sense in molecular biology. However, during our gene expression experiments using linear DNA in mammalian cells, we unexpectedly found that terminator can be placed in front of promoter. Correctly, terminator can be placed at another end from the end of coding sequence in linear DNA. If linear DNA is called from front to end along with the coding sequence direction, terminator is set at front, which we called ‘front terminator’ (Figure 3).
The front terminator gene construct is useful because the terminator::promoter segment can be used as a tool for the gene construction by the fusion with coding sequences. The terminator sequences can be attached in front of promoter and used to fuse with a coding sequence (Figure 3A). The front terminator constructs were used to analyze peroxisome targeting signal sequence (serine-lysine-leucine; SKL) (3). The mutant sequences were attached to the C-terminus of EGFP using oligonucleotide primers. The resulting PCR products were directly used for the transfection of HEK293 cells (Figure 3B).
Figure 3. Gene expression from front terminator construct. A) Front terminator-promoter fragment can be used for fusion PCR with a coding sequence of interest. The 5’-end of terminator and the 3’-end of coding sequence were connected with each other in mammalian cells after transfection through non-homologous end joining (NHEJ). B) Construction of EGFP containing C-terminal peroxisome targeting signal sequence (SKL: serine-lysine-leucine). For EGFP-SKL fusion protein expression, the CMV promoter (CMVp)-EGFP fragment was amplified with a terminator-attached forward primer and SKL attached reverse primer. EGFP was visualized by fluorescence microscopy at 24 h after transfection.
In our two papers (1, 3), we described three types of findings. First, the enhancer reagents, PEG and tRNA, allowed efficient transfection of mammalian cells in 96-well plates. Second, terminator primer was created based on the analysis of PCR-amplified DNA expression system. In linear DNA expression, terminator is exclusively required for the efficient expression. We assume that the end of mRNA, which is easily generated if there is an end of linear DNA, may be highly unstable without poly A sequence. The deletion analysis of PCR-amplified DNA revealed minimal functional sequences of SV40 and b-globin terminators, which developed the ‘terminator primers’. Third, gene manipulations with short terminator sequences lead us to produce the ‘front-terminator constructs’.
Gene expression even with the unusual location of terminator can be explained by non-homologous end joining (NHEJ) (Figure 3A). This is a DNA repair system commonly operated in eukaryotic organisms (4). We speculated that the 5’-end of terminator and the 3’-end of coding sequence were connected with each other after the front-terminator construct was introduced into mammalian cells (Figure 3A). End joining of the introduced DNA fragment was confirmed (3). Simply, in vivo DNA ligation was operated after the transfection of linear DNA. The application of NHEJ for gene manipulations in mammalian cells is innovative (3). However, we previously developed NHEJ-mediated gene manipulation methods in the yeast Kluyveromyces marxianus (5). Based on the studies on K. marxianus, we easily obtained the idea of NHEJ-mediated gene manipulations in mammalian cells. NHEJ is an evolutionarily conserved DNA repair mechanism. Therefore, the NHEJ-mediated gene manipulation will be applied to any of the other eukaryotic organisms except for the yeast Saccharomyces cerevisiae, a model organism with low NHEJ activity (6).
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Acknowledgements: This study was supported in part by JSPS KAKENHI (Grant No. 25660080), the Adaptable and Seamless Technology Transfer Program through Target-Driven R&D (JST, Japan), Mishima Kaiun Memorial Foundation, and the Yamaguchi University ’Pump-Priming Program’ for fostering research activities.
Mikiko Nakamura, Ph. D., Scientist
Present address: Ube Development Group, FUJIREBIO INC., 203-152 Yoshiwa Ushiake, Ube 759-0134, Japan,
Rinji Akada, Ph. D., Professor
Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Tokiwadai, Ube 755-8611, Japan