therapy is a novel field of research in the medical and biological sciences and has gradually developed in conjunction with the recent advances in recombinant DNA technology. The application of vectors is a crucial step in gene therapy. There are two types of vectors: integrated and non-integrated vectors. Integrated vectors facilitate the insertion of mutations, and may be responsible for diseases arising as a result of the position of the inserted gene (Buceta et al., 2011; Ley et al., 2013). Ideal non-integrated vectors present no potential risk in gene therapy, are stable through mitosis, and express high levels of the transgene (Girod et al., 2007). Matrix attachment region (MAR) (200-2000 bp), or the scaffold at- tachment region, is a DNA sequence rich in AT base pairs (>60%) attached to the matrix after enzymatic digestion.
The MAR provides a cis-acting element to the attached vector, avoids epigenetic gene silencing, and promotes the stability of mitotic chromosomes (Kamimura et al., 2011; Harraghy et al., 2011, 2012). The MAR sequence is a special DNA sequence that binds to the nuclear matrix. It is located in the bilateral non-coding regions of genes. MAR contains some characteristic sequence components, including a T-box (TTTTATTTTT), an A-box (AATAAAAA/CAA), an autonomously replicating DNA sequence, and fruit fly topoisomerase II recognition sites (Ehrhardt et al., 2008; Mucller and Flotte, 2008). The MAR also mediates the attachment of vectors to host cells (Chiaretti et al., 2008; Zheng et al., 2010; Calado et al., 2014). Nerve growth factor (NGF) is an important bioactive molecule in the nervous system.
NGF plays an important role in maintaining the growth and development of the nervous system and participates in the regeneration and repair of damaged nerves. Therefore, ex- erogenous NGF has been increasingly applied to the treatment of cerebral injuries in clinical settings. However, NGF cannot easily penetrate the blood-brain barrier because of its large molecular weight. Additionally, the intravenous administration of NGF does not allow for optimum efficiency. In addition to its high cost, these factors limit the application of NGF to the treatment of disorders and defects in the central nervous system (Haase et al., 2010; Liu et al., 2013). Gene transfection is a novel tool that can be used in the application of multiple- neurotrophic factors. Preliminary studies have shown that the MAR characteristic element in human interferon-β can mediate the adhesion of vectors to Chinese hamster ovary (CHO) cells.
However, its possible effect on enhancing the transgene expression remains to be de- termined. MAR characteristic motifs, such as the AT sequences in interferon-β (2200 bp, Gen – Bank: M83137.1), were cut and spliced to short MAR characteristic sequences (367 bp). MAR was amplified by polymerase chain reaction (PCR), and directed cloning was performed using the Kpn I and Bam HI enzyme restriction sites generated in the 5′-terminus of the primers. The PCR conditions were as follows: 95°C for 3 min, 94°C for 40 s, 60-56°C for 30 s, and 72°C for 40 s. Each annealing temperature was used for 4 cycles. Finally, the PCR product was amplified using an annealing temperature of 55°C for 20 cycles. A final extension step was performed at 72°C for 3 min. The T-vector (TaKaRa Bio, Inc., Dalian, China), connected to the MAR fragment and the pEGFP-C1 vector, was digested using the corresponding restriction enzymes. The intermediate vector, pEGFP-C1-MAR, was then constructed. Specific NGF primers were designed for the NGF gene (GenBank: AF150960.1). DNA from human peripheral blood was used as the template to amplify NGF. Hin dIII and Kpn I restriction sites was introduced.
pEGFP-C1 and pEGFP-C1-MAR vectors were amplified by PCR, and the NGF was directionally inserted upstream of MAR using the multiple-cloning site of the pEGFP-C1 vector. The pEGFP-C1-NGF and pEGFP-C1-MAR-NGF were then constructed, and used for further digestion and sequencing to verify the correctness of the sequences (Figure 1). CHO cells (Institute of Laboratory Animal Sciences, Beijing, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA), supplemented with 10% inactivated fetal calf serum (Gibco) on a 6-well plate (3 x 10 6 cells/ well) at 37°C and 5% CO 2 . Three transfection groups were prepared: the pEGFP-C1-NGF vector transfection group, pEGFP-C1-MAR-NGF vector transfection group, and pEGFP- C1 negative control group. The transfection was performed using the Summa-so fast gene transfection reagent kit (Summa Biotechnology Co., Ltd., Xiamen, China) according to the manufacturer’s protocols. G418 (Calbiochem, La Jolla, CA, USA; final concentration: 800 μg/mL) was used 24 h after transfection to select the transfected cells for 2 weeks. The culture medium was changed every 2-3 days.
Stably transformed cell colonies were trypsinized (0.25% trypsin). Transfected cells in each group were selected by the dilution method, in order to obtain the monoclonal cell lines. These cells were then split onto a 96-well culture plate and transferred to culture flasks after 7 days. The cells were analyzed at a cell density of up to 80-90%. The collected cells were adjusted to the same concentration in each group (1 x 10 6 /mL), and the same amount of cells was collected (via centrifugation) for each group. The total RNA was extracted from these cells using the Total RNA Isolation kit (TaKaRa Bio Inc.). RNA was reverse transcribed to cDNA using the RT-PCR Kit (TaKaRa Bio Inc.). The primers designed based on the NGF gene were P1: 5′-ATGTCCATGTTGTTCTACACTCT-3′ and P2: 5′-TCAGGCTCTTCTCACAGCCTTCCT-3′.
The PCR product was 726 bp long. The GAPDH control primers were P3: 5′-CACATATTCTGGAGGAGCCTCC-3′ and P4: 5′-ACGGTGCCATGGAATTTGCCAT-3′. The PCR product was 295 bp long. These primers were synthesized by Sangon Biotechnology Development Co., Ltd. (Shanghai, China). The target and internal reference genes were amplified using the reverse-transcribed cDNA. The PCR conditions were as follows: initial denaturation at 95°C for 5 min; 30 cycles of 94°C for 40 s, 55°C for 40 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. The amplified PCR products were separated by electrophoresis, using a 2% agarose gel.
The target bands were imaged using a UV analyzer, and the Bander leader 3.0 gel image processing software was used to analyze the image. The fold-change of the target gene over the GAPDH gene reflected the relative expression level of target mRNA. The DMEM culture was replaced when a cell density of 80-90% was attained. The cell culture supernatant was collected. The cells were counted after 24 h. The NGF secretory protein concentration in the cell culture supernatant was measured using an NGF enzyme-linked immunosorbent assay kit (DingGuo Biological Technology Co., Ltd., Beijing, China). The experiment was repeated three times. The attached vectors were isolated from the CHO cells using the Hirt (1967) cracking method and subsequently transformed. Positive transformants were selected and cultured in tubes with the LK liquid medium overnight at 37°C on a shaking table. The plasmids were isolated from the cells using the plasmid isolation kit (Solarbio, Beijing, China) according to the manufacturer’s protocols.
The plasmids were digested with Kpn I and Bam HI or Hin dIII and Kpn I, and subsequently digested with Kpn I. All data were statistically analyzed using the SPSS v.18.0 software platform (SPSS Inc., Chicago, IL, USA). All data are reported as means ± standard deviation. The differences among different groups were estimated by ANOVA. P values <0.05 were considered to be statistically significant. The constructed vectors were digested using the different enzymes. Agarose gel electrophoresis indicated the presence of a 726-bp fragment in pEGFP-C1-NGF when digested with Hin dIII and Kpn I; however, a linear DNA fragment was obtained after digestion with Kpn I. These findings demonstrated the successful construction of the pEGFP-C1-NGF vector. The pEGFP-C1-MAR-NGF vector was digested using Kpn I and Bam HI, as well as Hin dIII and Kpn I. Two fragments were obtained (367 and 726 bp, respectively).
A linear DNA fragment was upon digestion with Kpn I. The results were consistent with the expected fragment sizes. Sequencing results also demonstrated the successful construction of the pEGFP-C1- NGF and pEGFP-C1-MAR-NGF expression vectors. A 726-kb band, specific for the NGF mRNA, was detected. The results of the image analysis showed that the CHO cells transfected with the pEGFP-C1 plasmid expressed low levels of NGF (0.15 ± 0.02), and a weak (brightness) target band. The expression of NGF mRNA in the CHO cells transfected with the pEGFP-C1-MAR-NGF plasmid was significantly higher (0.25 ± 0.03) than that in CHO cells transfected with the pEGFP-C1-NGF (0.47 ± 0.03) plasmid. NGF expression in both cell lines was significantly higher than that in normal CHO cells (Figure 2; P < 0.05).