Effects of different 2A peptides on transgene expression mediated by tricistronic vectors in transfected CHO cells.

Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.

Two human MARs effectively increase transgene expression in transfected CHO cells.

Matrix attachment regions (MARs) can enhance the expression level of transgene in Chinese hamster ovaries (CHO) cell expression system. However, improvements in function and analyses of the mechanism remains unclear. In this study, we screened two new and more functional MAR elements from the human genome DNA. The human MAR-3 and MAR-7 element were cloned and inserted downstream of the polyA site in a eukaryotic vector. The constructs were transfected into CHO cells, and screened under G418 to produce the stably transfected cell pools. The expression levels and stability of enhanced green fluorescent protein (eGFP) were detected by flow cytometry. The transgene copy number and transgene expression at mRNA level were detected by quantitative real-time PCR. The results showed that the expression level of eGFP of cells transfected with MAR-containing vectors were all higher than those of the vectors without MARs under transient and stably transfection. The enhancing effect of MAR-7 was higher than that of MAR-3. Additionally, we found that MAR significantly increased eGFP copy numbers and eGFP gene mRNA expression level as compared with the vector without. In conclusion, MAR-3 and MAR-7 gene can promote the expression of transgene in transfected CHO cells, and its effect may be related to the increase of the number of copies.

Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells.

Nonviral episomal vectors present attractive alternative vehicles for gene therapy applications. Previously, we have established a new type of nonviral episomal vector-mediated by the characteristic motifs of matrix attachment regions (MARs), which is driven by the cytomegalovirus (CMV) promoter. However, the CMV promoter is intrinsically susceptible to silencing, resulting in declined productivity during long-term culture. In this study, Chinese hamster ovary (CHO) cells and DNA methyltransferase-deficient (Dnmt3a-deficient) CHO cells were transfected with plasmid-mediated by MAR, or CHO cells were treated with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Flow cytometry, plasmid rescue experiments, fluorescence in-situ hybridization (FISH), and bisulfite sequencing were performed to observe transgene expression, its state of existence, and the CpG methylation level of the CMV promoter. The results indicated that all DNA methylation inhibitor and methyltransferase deficient cells could increase transgene expression levels and stability in the presence or absence of selection pressure after a 60-generation culture. Plasmid rescue assay and FISH analysis showed that the vector still existed episomally after long-time culture. Moreover, a relatively lower CMV promoter methylation level was observed in Dnmt3a-deficient cell lines and CHO cells treated with 5-Aza-2'-deoxycytidine. In addition, Dnmt3a-deficient cells were superior to the DNA methylation inhibitor treatment regarding the transgene expression and long-term stability. Our study provides the first evidence that lower DNA methyltransferase can enhance expression level and stability of transgenes mediated by episomal vectors in transfected CHO cells.

Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells.

Low-level and unstable transgene expression are common issues using the CHO cell expression system. Matrix attachment regions (MARs) enhance transgene expression levels, but additional research is needed to improve their function and to determine their mechanism of action. MAR-6 from CHO chromosomes actively mediates high and consistent gene expression. In this study, we compared the effects of two new MARs and MAR-6 on transgene expression in recombinant CHO cells and found one potent MAR element that can significantly increase transgene expression. Two MARs, including the human CSP-B MAR element and DHFR intron MAR element from CHO cells, were cloned and inserted downstream of the poly(A) site in a eukaryotic vector. The constructs were transfected into CHO cells, and the expression levels and stability of eGFP were detected by flow cytometry. The three MAR sequences can be ranked in terms of overall eGFP expression, in decreasing order, as follows: human CSP-B, DHFR intron MAR element and MAR-6. Additionally, as expected, the three MAR-containing vectors showed higher transfection efficiencies and transient transgene expression in comparison with those of the non-MAR-containing vector. Bioinformatics analysis indicated that the NFAT and VIBP elements within MAR sequences may contribute to the enhancement of eGFP expression. In conclusion, the human CSP-B MAR element can improve transgene expression and its effects may be related to the NFAT and VIBP elements.

[Effects of Different Promoters and MAR Combinations on Transgene Expression of Recombinant CHO Cells].

OBJECTIVE: To analyze the effects of different promoters and matrix attachment region (MAR) on the expression of transgene in Chinese hamster ovary (CHO) cells. METHODS: The expression vector was constructed by the combination of beta globin MAR (gMAR) with the human cytomegalovirus immediate-early promoter (CMV-IE) and simian virus 40 (SV40) promoter. These vectors were transfected into CHO cells,after 48 h,the transient expression of enhanced green fluorescent protein (eGFP) was observed; G418 was used to screen stably transformed cell lines,and the expression level of eGFP in CHO cells was analyzed by flow cytometry. The relative copy numbers of eGFP were analyzed by qPCR. RESULTS: Without gMAR expression vector,the expression of eGFP which was driven by CMV-IE promoter was stronger than that of SV40 promoter; gMAR could increase the expression level of eGFP driven by CMV-IE promoter,but did not show any enhancement in SV40 promoter. The expression level of eGFP which containing gMAR on both sides was stronger than that of gMAR on one side driven by CMV-IE promoter; After G418 screening,the expression level of eGFP containing gMAR driven by SV40 promoter wasunstable,the fluorescence gradually weakened,therefore,we only analyzed the expression vector stably expressing the eGFP gene driven by CMV-IE promoter by flow cytometry and qPCR. Compared with the expression vector without gMAR containing CMV-IE promoter,flow cytometry showed that the expression levels of eGFP on one and both sides with gMAR were increased by 9.85-fold and 12.94-fold,respectivley; The result of qPCR showed that the copy number of the eGFP gene without gMAR was set to 1,the copy number of the eGFP gene in the expression vector driven by CMV-IE with gMAR on one side and both sides were 3.68-fold and 9.25-fold,respectively. CONCLUSION: The activity of CMV-IE promoter is stronger than that of SV40 promoter. gMAR can enhance the expression levels of transgene,which may be related to the increase of gene copy number.

[Effect of Intron Orientation on the Expression of Transgene Imposed by MAR Expression Vector in Stably Recombinant CHO Cells].

OBJECTIVE: To determine the effect of intron orientation on the transgene expression level imposed by matrix attachment region (MAR) expression vector. METHODS: The MAR of ß-globin was amplified by PCR, and then cloned into MAR expression vectors. An intron sequence was digested with restriction enzyme, ligated to the MAR expression vector in reverse orientation, and then transfected into Chinese hamster ovary (CHO) cells. The transfected stable cells were screened by G418. The level of chloramphenicol acetyltransferase (CAT) gene expression was analyzed by ELISA method. RESULTS: The transgene expression levels of CHO cells with the two expression vectors with a positive intron or without MAR were higher than that of CHO cells with an expression vector with reverse intron (P < 0.05). MAR did not improve transgene expression with reverse intron presence. CONCLUSION: Different orientation of intron can affect transgene expression in recombinant CHO cells. The transgene expression level can be increased using positive intron and MAR.

The effect of recombinant lentiviral vector encoding miR-145 on human esophageal cancer cells.

miR-145, a newly identified microRNA molecule, is hypothesized to function as a tumor suppressor, but this activity has not been investigated in esophageal l carcinoma (EC). The aim of this study was to investigate the effect of miR-145 on the biological features of EC cells. miR-145 was obtained using PCR technology and cloned into the lentiviral vector, pLVX-IRES-ZsGreen1, to construct the resulting vector, pLVX-IZ-miR-145. The vector was packaged, the viral titer was tested, and ECA109 cells were infected with the optimal viral titer. Cells that were stably transfected with miR-145 were screened. Flow cytometry was used to analyze enhanced green fluorescence protein gene expression, and to measure cell apoptosis and cell cycle. miR-145 expression was detected by real-time fluorescent quantitative PCR. Furthermore, cell proliferation was assayed using CCK-8 assay. The pLVX-IZ-miR-145 vector was successfully constructed, and the viral titer achieved up to 5.0 × 10(8) TU/mL. The transfection efficiency was 90 %. Compared to the control group, the expression level of miR-145 in the transfected group was significantly higher (185-fold, P < 0.05). miR-145 overexpression significantly inhibited esophageal cancer cell proliferation (P < 0.05). Moreover, the number of cells at the G2/M stage, as well as the cell apoptotic rate, in the miR-145-transfected group was significantly increased (P < 0.05). Our study reveals that overexpression of miR-145 inhibits cell proliferation, increases apoptosis, and influences the cell cycle progression of EC cell.

Distance effect of matrix attachment regions on transgene expression in stably transfected Chinese hamster ovary cells.

The ß-globin matrix attachment regions (MARs) were inserted into the 5'-site of the eukaryotic expression vector cassette and DNA fragments 350 and 750 bp in length were inserted into the site to generate expression vectors with varying distances between the expression cassette and MAR. The vectors containing MARs increased chloramphenicol acetyltransferase (CAT) expression levels compared to the negative control vector lacking the MAR; the highest expression increase was 3.8-fold. A greater MAR-transgene distance (750 bp) correlated with a greater increase in transgene expression when compared to the control vector that lacked separation between the MAR and transgene. CAT gene copy numbers were higher in cells transformed with the vector possessing a smaller MAR-transgene distance (350 bp) than in cells belonging to the other three groups. However, MAR-induced transgene expression levels did not exhibit a direct relationship with gene copy number.

[Percutaneous pelvic fracture stabilization using CT-based 3D navigation software combined with targeting mechanical arm: a cadaver model trial].

OBJECTIVES: To investigate a new targeting mechanical arm for CT-based navigated percutaneous fixation of pelvic fractures, and to evaluate the safety and efficiency of the procedures. METHODS: Using CT-based 3D navigation software combined with targeting mechanical arm, percutaneous insertion of pelvic models (3 dry human cadaver pelvic skeletons and 5 plastic Sybone pelvic models) were performed, 8 pelvic models allowed percutaneous cannulated screw insertion of both S-I joint (2 S-I screws placement for each side, total 32 screws in this experiment) and both superior ramus (1 ramus medullary screw placement for each side, total 16 screws in this experiment). Percutaneous insertion of pelvic models (4 dry human cadaver pelvic skeletons and 4 plastic Sybone pelvic models, 1 S-I screws and 1 ramus medullary scre placement for each side, 32 screws in this experiment) were performed using fluoro-navigation system (Stryker, USA). Time necessary for every screw insertion were recorded. Accuracy of screw placement was assessed using C-arm imaging and direct eyes inspecting. The time and accuracy of the two methods were compared. RESULTS: The time required for the CT-based 3D navigation procedure (3.6 ± 1.2) min was significantly less than using the targeting mechanical arm compared to drilling freehand with navigation (9.1 ± 0.8) min (t = 2.50, P < 0.01). There was no significant difference in accuracy between the two methods. CONCLUSION: CT-based 3D navigation software combined with targeting mechanical arm should be potential to apply percutaneous sacroiliac screwing for pelvic fractures with more accurate and more reliable.

Percutaneous Robot-Assisted versus Freehand S2 Iliosacral Screw Fixation in Unstable Posterior Pelvic Ring Fracture.

OBJECTIVES: To assess the efficiency, safety, and accuracy of S2 (IS) screw fixation using a robot-assisted method compared with a freehand method. METHODS: This is a retrospective clinical study. We analyzed the patients treated with S2 IS screw fixation for unstable pelvic fractures from January 2016 to January 2019 in our institution. Sixty-three patients (17 men and 46 women) aged between 21 and 55 years (with an average age of 39.22 ± 9.28) were included in this study. According to the Tile classification, there were 26 (41.3%) type B fractures and 37 (58.7%) type C fractures. All patients were divided into robot-assisted (RA) group (38 patients) or the traditional freehand (FH) group (25 patients). In RA group, the S2 IS screws were implanted with a robot-assisted technique. And S2 IS screws were implanted with a traditional freehand technique in FH group. The screw-related complications were recorded during and after the surgery. The position of all screws and fracture reduction was assessed by postoperative CT scans according to the Gras classification. The number of guide wire attempts and the radiation exposure for S2 screw implantation during operation were also recorded. Finally, the Matta standard was used to evaluate the fracture reduction of the IS joint. RESULTS: A total of 89 IS screws were implanted into S2 iliosacral joint. Fifty-four screws were placed by RA (38 patients) and 35 screws were by FH (25 patients). There was no difference between the two groups with respect to demographic data. There was no screw-related complications or revision surgery in any group. In terms of screw placement, the excellent and good rate was 100% in the RA group, better than that in the FH group where it was only 85.7% (P < 0.001). The fluoroscopy time was 8.06 ± 3.54 s in RA group, which was much less than that in the FH group (27.37 ± 8.82 s, P < 0.001). The guide wire attempts in the RA group (0.685 ± 0.820) were much less than those in the FH group (5.77 ± 3.34) (P < 0.001). Both the fluoroscopy time per screw and the number of guide wire attempts in the RA group were much less than those in the FH group (P < 0.001). The overall postoperative excellent and good rate of Matta standard in RA and FH groups were 86.8% (34/4) and 90.0% (23/25), respectively (P = 0.750), and there was no statistical difference. CONCLUSION: The robot-assisted surgery is an accurate and minimally invasive technique. S2 IS screw implantation assisted by TiRobot to treat the posterior pelvic ring fractures, have a high success rate than the freehand technique. Percutaneous RA S2 IS screw fixation for unstable posterior pelvic ring injuries is safe and clinically feasible and has great clinical application value.

The CAG promoter maintains high-level transgene expression in HEK293 cells.

The vast majority of therapeutic recombinant proteins are produced in mammalian cell lines. However, proteins generated in nonhuman cell lines, such as Chinese hamster ovary (CHO) cells, are decorated with human-like glycan structures that differ from those of human cells, and these may induce immunogenic responses in human cells. Human embryonic kidney cells (HEK293F) are also extensively used as hosts for the expression of recombinant therapeutic proteins, but their utility is limited by the low expression of transgenes in these cells. Here, we investigated recombinant protein expression from eight frequently used promoters in transfected HEK293F cells. The expression levels and stability of the transgenes were evaluated by flow cytometry and qRT-PCR. The most efficient expression (in terms of both mRNA and protein yields) was achieved using a cytomegalovirus (CMV) major immediate-early enhancer combined with the chicken beta-actin promoter (CAG) promoter, as compared to all other tested promoters under both transient and stable transfection conditions. In addition, application of mild hypothermia (i.e., 33 °C) after transfection improved the positive effect of the CMV enhancer fused to the chicken beta-actin promoter (CAG promoter) on enhanced green fluorescent protein (eGFP) expression. Although the temperature sensitivity of the CMV promoter is greater than that of CAG promoter, recombinant protein levels were still highest when expression was driven by the CAG promoter. When eGFP was replaced with hepatitis B surface antigen, the CAG promoter still showed the highest transgene expression. In conclusion, our data show that the CAG promoter is a strong promoter for recombinant protein expression in HEK293F cells.

[Primary clinical application of traction reductor for lower limb fracture].

OBJECTIVE: To develop a traction reductor for the reduction of lower limb fractures during the minimally invasive surgery and explore its safety and efficacy. METHODS: From February 2007 to March 2009, closed or limited open reduction plus percutaneous plate and screw internal-fixation were conducted in 34 patients with fracture of distal femur and tibia metaphysic, among which there were 3 distal femoral fractures (2 33-B, 1 33-C), 14 proximal tibial fractures (9 41-A, 3 41-B, 2 41-C) and 17 distal tibial fractures (9 43-A, 5 43-B, 3 43-C, 2 Gustilo I a), according to the Association for Osteosynthesis-Orthopaedic Trauma Association (AO-OTA) classification. Besides, closed reduction plus interlocking intramedullary nailing on tibial shaft fracture were applied in 36 patients (7 42-A, 21 42-B, 8 42-C, 2 Gustilo I a). All the 70 patients, with an average age of 37.6 years (range: 17 to 63 years) and average time before surgery of 4.7 d (range: 0.7 to 12.0 d), underwent reduction by self-designed traction reductor for lower limb fracture in the surgery. The reduction duration and C-arm fluoroscopy time were recorded. Recovery of the force line of affected limbs after surgery was determined by whether the line from anterior superior iliac spine to the interdigit between the first and second toe-web passed the patella center. And the distance from bilateral anterior superior iliac spine to medial malleolus tip as well as the difference between lower limbs were recorded to determine the recovery of length after surgery. Meanwhile, the varus-valgus and anteroposterior angulations after reduction were measured by AP and lateral X-ray. RESULTS: The reduction duration was 12.7 min (range: 7 to 31 min); X-ray fluoroscopy time, 1.3 min (range: 0.4 to 3.0 min); length difference between both lower limbs (6.5 ± 1.1) mm; and axial alignment difference (7.0 ± 1.8) mm. The X-ray result showed that varus-valgus angle was (2.75 ± 0.16)°; and anteroposterior angulation (5.13 ± 0.51)°. CONCLUSION: The traction reductor for lower limb fracture could achieve satisfying fracture reduction in the minimally invasive surgery of distal femur, tibia metaphysic and tibial shaft fracture.

Guidance for Treatment of Pelvic Acetabular Injuries with Precise Minimally Invasive Internal Fixation Based on the Orthopaedic Surgery Robot Positioning System.

Pelvic acetabular fracture is a common kind of fracture, mostly caused by high energy injuries. It is associated with high mortality and disability rates. The aim of surgical treatment of pelvic acetabular fractures is to restore the symmetry and stability of the pelvic ring structure and the anatomical structure of acetabular. Open reduction internal fixation is often used for the treatment of such fractures, but open surgery is in cases of serious injury, more bleeding, and high risk of infection. With the development of minimally invasive technology and the concept of the bone channel structure, the percutaneous lag screw technique for the treatment of pelvic and acetabular fractures has been applied in clinical practice and has proven to be effective. However, the anatomical structure of the pelvis and acetabulum is complex, and there are many important nerves and vessels adjacent to it. Traditional fluoroscopy screw placement is prone to screw malposition, and even minor angle changes may lead to screw perforation and damage of nerve vessels. The problem of radiation exposure is also noteworthy. Robotic-assisted surgery can be used to carry out screw position planning through preoperative imaging, intraoperative real-time tracking, and mechanical arm assistance to ensure that the screw placement position is consistent with the planning. In this way, robotic-assisted surgery can be used to accurately insert lag screws, and can reduce surgical risk and radiation exposure. This guide uses the TiRobot system as an example to describe the application of robot surgery in detail, aiming at standardizing the application of robots in orthopaedic surgery.

Evaluation of a Bi-Planar Robot Navigation System for Insertion of Cannulated Screws in Femoral Neck Fractures.

OBJECTIVE: To evaluate the bi-planar robot navigation system for insertion of cannulated screws in femoral neck fractures. METHOD: Between January 2016 and December 2016, 60 patients with femoral neck fractures were separately treated using percutaneous cannulated screws assisted by the bi-planar robot navigation system (robot group) and conventional freehand surgery (freehand group). The fluoroscopy time, the number of drilling attempts, and the operation time were recorded during operations; the dispersion and parallelism of the cannulated screws on the posteroanterior and lateral images were measured after operations. Patients were followed up for 12-24 months and the Harris scores and the final results of the two groups were compared. RESULTS: During bi-planar robot navigation system-assisted surgery, the fluoroscopy time for acquisition of images was 2.3 seconds on average, and the time for planning screws during the operation was 2.8 min on average. The average fluoroscopy time during the placement of the guide pin was 5.7 seconds and 14.14 seconds (P = 0.00), respectively. The average time of the placement of the cannulated screws was 12.7 min and 19.4 min (P = 0.00), respectively, in the robot group and the freehand group. In the robot group, only one guide pin was replaced during the operation, and the average number of adjustments for each guide pin was 2.39 in the freehand group. The screw parallelism and dispersion measured by postoperative imaging in the robot group were significantly superior to those in the freehand group. From postoperative CT it was evident that there were 5 cases of screws exiting the posterior cortex in both groups. During the follow-up phase, 1 case of femoral head necrosis and 5 cases of femoral neck shortening of more than 10 mm occurred in the robotic navigation group; 3 cases of femoral head necrosis, 1 case of fracture nonunion, and 2 cases of shortening of more than 10 mm occurred in the freehand group. At 18 months after surgery, the average Harris scores of the patients were 85.20 and 83.45, respectively, with no significant difference. CONCLUSION: Using bi-planar robot navigation system-assisted placement of femoral neck cannulated screws can significantly reduce the time of intraoperative fluoroscopy, drilling attempts, and operation time. The placed screws are superior to the screws placed freehand in relation to parallelism and dispersion. However, it is still necessary for surgeons to have a good reduction of the femoral neck fracture before surgery and to be proficient in the operation of the robot navigation system. In summary, the bi-planar robot navigation system is an effective assistant instrument for surgery.

[Percutaneous screw fixation in treatment of fractures of acetabular columns using computer-assisted imaging navigation system: experiment with cadaver model].

OBJECTIVE: To establish a new imaging protocol to acquire the most appropriate fluoro-images for fluoro-navigated percutaneous fixation of acetabular fracture and to evaluate the safety and efficiency of the procedures. METHODS: Guide needles were inserted into the bilateral anterior and posterior columns of the acetabula of 4 dry human cadaver pelvic skeletons and 4 plastic pelvic models. Then the pelvis skeletons were fixed to imaging guided surgery mimic operation modules. Dynamic fluoroscopy was conducted with C-arm X-ray machine vertically on the 4 virtual planes of the acetabulum, inner, outer, anterior, and posterior from multiple angles. The fluoroscopic images clearly showing the guide needles, anterior and posterior columns, and acetabulum were selected as registration images, and the relative space positions between the C-arm X-ray fluoroscope and pelvis and operation table. Guided by the navigation system, totally 16 titanium hollow screws were inserted into bilateral anterior and posterior columns of acetabula of the 4 pelvis skeletons. The screw positions were estimated by visual method. The time needed to position the C-arm so as to obtain the standard registration image, time needed for fluoroscopy, and operation time, including establishment of navigation system, software interface operation, and screw insertion, were recorded. RESULTS: All the screws were inserted to the satisfying positions: placed within the desired bony corridor of the column and none of then were inserted into the joint. While inserting the screw into the anterior column fluoroscopy should be conducted with obturated oblique view, obturated inlet view, obturated oblique outlet view, or pelvic AP view. While inserting the screw into the posterior column fluoroscopy should be conducted with iliac-oblique view, obturated oblique view, pelvic inlet view, or obturated oblique outlet view. The total surgical time required for screw insertion was 11.7 min for anterior column, and was 9.2 min for posterior column. 9.5 and 7.3 minutes were needed to position the C-arm X-ray machine so as to obtain the images of the anterior and posterior columns respectively. 2.9 and 1.7 seconds were needed for the actual fluoroscopy during insertion of the screws into the anterior and posterior columns respectively. CONCLUSION: The standard registered fluoro-images are different from the conventional standard Judet-Letournel oblique views. How to get appropriate fluoro-images is the key point for fluoro-navigated percutaneous fixation of acetabular fractures.

Retraction Note: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells.

This paper has been retracted at the request of the authors.

[Accurate traction of long bone fracture with full-length planning module of orthopedic robot system: experiments in vitro and in vivo].

OBJECTIVE: To evaluate the precision in location and clinical flexibility of the newly designed full-length planning module of orthopedic robot system in treatment of fractures of long bone. METHODS: Nine plastic tibia models were selected for the image mosaicing. The full length of each tibia model was measured on the constructed panorama and compared with the real model length to record the length deviation and conduct the precision analysis. Fracture of tibia and fibula with shortening and angulation deformity was caused on a cadaver specimen with two lower limbs. Full-length planning was carried out on the entire tibial panorama with the fracture. After the reduction distance was determined quantitatively, automatic close traction procedure was carried out with the tibial reduction frame to analyze the precision and effectiveness of this module. At the same time, the relative length variation between the two bone fragments was monitored utilizing video camera to ensure the safety of the reduction operation. Image mosaicing, surgical planning, and bone traction were performed on a clinical case of tibial fracture to validate the clinical feasibility of the module. RESULTS: An entire tibial panorama could be constructed from 7 - 10 C-arm images collected during the operation. 1.5 min was needed for image collection. The average mosaicing and planning time was 3 min. The mosaicing error was less than 1.5 mm. The average time for the traction frame installation and traction operation was 4 min. Traction resulted in accurate reposition of the fracture ends meeting the requirement of surgery in both the cadaver specimen and the clinical case. CONCLUSION: The newly designed full-length planning module of orthopedic robot system is easy to use and provides effective and accurate traction result in long bone fracture therapy. This module can not only achieve the minimally invasive surgery, but also dramatically decrease the radiation damage to the medical staff.

Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells.

In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.

Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells.

Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human ß-interferon and ß-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes.

Computer-assisted auto-frame navigation system for distal locking of tibial intramedullary nails: a preliminary report on clinical application.

OBJECTIVE: To evaluate the clinical feasibility and effect of the computer-assisted auto-frame navigation system for distal locking of tibial intramedullary nails. METHODS: The hardware components of the system included a PC computer with a monitor, auto mechanical stereotactical localization cubic frame, foot holder and localization operative apparatus. Special navigation software can be used for registration of X-ray fluoroscopic images and real-time controlling navigation of tools. Twenty-one cases of close tibial and fibular fractures were treated with closed intramedullary nailing, 6 of which involved in middle third, 12 in middle and lower third, 3 in lower third. C-arm alignment and registration time, fluoroscopic time and drilling time involved in the locking procedure were recorded. The size of unreamed or reamed tibial nails ranged from 8/300-11/330. RESULTS: All distal holes except 1 were locked successfully. In 9 of 41 locked holes (21.95%), the drill bit touched the canal of locking hole without damage of the nail and clinical consequences. The fluoroscopy time per pair of screws was 2.23+/-0.31 s. CONCLUSIONS: The computer-assisted auto-frame navigation system for distal locking is well designed, easy to operate and do not need additional instruments during the procedure. The developed system enables the physician to precisely navigate surgical instruments throughout the anatomy using just a few computer-calibrated radiographic images. The total time of X-ray exposure per procedure can be significantly reduced.