SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells have become the most widely utilized mammalian cell line for the production of recombinant proteins. However, the product yield and transgene instability need to be further increased and solved. In this study, we investigated the effect of five different introns on transgene expression in CHO cells. hCMV intron A, adenovirus tripartite leader sequence intron, SV40 intron, Chinese hamster EF-1alpha gene intron 1 and intervening sequence intron were cloned downstream of the eGFP expression cassette in a eukaryotic vector, which was then transfected into CHO cells. qRT-PCR and flow cytometry were used to explore eGFP expression levels. And gene copy number was also detected by qPCR, respectively. Furthermore, the erythropoietin (EPO) protein was used to test the selected more strong intron. The results showed that SV40 intron exhibited the highest transgene expression level among the five compared intron elements under transient and stable transfections. In addition, the SV40 intron element can increase the ratio of positive colonies and decrease the coefficient of variation in transgene expression level. Moreover, the transgene expression level was not related to the gene copy number in stable transfected CHO cells. Also, the SV40 intron induced higher level of EPO expression than IVS intron in transfected CHO cell. In conclusion, SV40 intron is a potent strong intron element that increases transgene expression, which can readily be used to more efficient transgenic protein production in 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.

Construction strategies for developing expression vectors for recombinant monoclonal antibody production in CHO cells.

Recent years have seen the use of recombinant proteins in the treatment of different diseases. Among them, monoclonal antibodies (mAbs) are currently the fastest growing class of bio-therapeutic recombinant proteins. Chinese hamster ovary (CHO) cells are the most commonly used host cells for production of these recombinant mAbs. Expression vectors determine the expression level and quality of recombinant mAbs. Currently, few construction strategies for recombinant mAbs expression vectors in CHO cells have been developed, including monocistronic vector, multiple-promoter expression vector, and tricistronic vector mediated by internal ribosome entry site (IRES) or Furin-2A element. Among them, Furin-2A-mediated vector is an effective approach due to advantages of high "self-cleavage" efficiency, and equal expression of light and heavy chains from a single open reading frame. Here, we have reviewed the progress in development of different strategies for constructing recombinant mAb expression vectors in CHO cells and its potential advantages and disadvantages.

[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.

The EF-1α promoter maintains high-level transgene expression from episomal vectors in transfected CHO-K1 cells.

In our previous study, we demonstrated that episomal vectors based on the characteristic sequence of matrix attachment regions (MARs) and containing the cytomegalovirus (CMV) promoter allow transgenes to be maintained episomally in Chinese hamster ovary (CHO) cells. However, the transgene expression was unstable and the number of copies was low. In this study, we focused on enhancers, various promoters and promoter variants that could improve the transgene expression stability, expression magnitude (level) and the copy number of a MAR-based episomal vector in CHO-K1 cells. In comparison with the CMV promoter, the eukaryotic translation elongation factor 1 α (EF-1α, gene symbol EEF1A1) promoter increased the transfection efficiency, the transgene expression, the proportion of expression-positive clones and the copy number of the episomal vector in long-term culture. By contrast, no significant positive effects were observed with an enhancer, CMV promoter variants or CAG promoter in the episomal vector in long-term culture. Moreover, the high-expression clones harbouring the EF-1α promoter tended to be more stable in long-term culture, even in the absence of selection pressure. According to these findings, we concluded that the EF-1α promoter is a potent regulatory sequence for episomal vectors because it maintains high transgene expression, transgene stability and copy number. These results provide valuable information on improvement of transgene stability and the copy number of episomal vectors.

Arthroscopic debridement improves range of motion for heterotopic ossification after total knee replacement: a retrospective cohort study.

The presence of heterotopic ossification (HO) after primary total knee replacement (TKR) is rare and associated with limited mobility and stiffness of the knee. This study aimed to identify if the arthroscopic debridement after TKR could decrease HO and improve the function and range of motion. Thirty HO patients after TKR were retrospectively separated into 2 cohorts. 15 patients of group A accepted the arthroscopic debridement, while 15 patients of group B only had non-operative treatment, mainly including oral nonsteroidal anti-inflammatory drugs (NSAIDs) and rehabilitative treatment. Visual analog scale (VAS) scores, knee society knee scores (KSS), range of motion (knee flexion and knee extension) were obtained before treatment and at 1 month, 3 months, and 6 months after treatment. Radiography of after-treatment was also evaluated to assess the changes in HO. There were 3 males and 27 females with a mean age of 67.4 ± 0.8 years in group A and 68.2 ± 1.3 in group B. The onset time of HO was 3-6 months. The maximum size of the ossification was < 2 cm in 23 knees, 2 cm < heterotopic bone < 5 cm in 6 knees and > 5 cm in 1 knee. The size of HO decreased gradually in all knees by X-ray film at the last follow-up. There were no significant differences in VAS scores after replacement between two groups (p > 0.05). The average range of motion preoperatively in group A was - 15.2-90.6°, which postoperatively increased to - 4.2-110.0°. Meanwhile, the KSS scores and average range of motion of the group A were better than those of the group B at each follow-up time after treatment. Arthroscopic debridement can decrease HO seen from postoperative X-rays, improve the function and range of motion, as well as the pain remission between two groups are comparable. Consequently, arthroscopic resection of HO after TKR is recommended as soon as there is aggravating joint stiffness.

TMBIM1 is an inhibitor of adipogenesis and its depletion promotes adipocyte hyperplasia and improves obesity-related metabolic disease.

Obesity is characterized by the excessive accumulation of the white adipose tissue (WAT), but healthy expansion of WAT via adipocyte hyperplasia can offset the negative metabolic effects of obesity. Thus, identification of novel adipogenesis regulators that promote hyperplasia may lead to effective therapies for obesity-induced metabolic disorders. Using transcriptomic approaches, we identified transmembrane BAX inhibitor motif-containing 1 (TMBIM1) as an inhibitor of adipogenesis. Gain or loss of function of TMBIM1 in preadipocytes inhibited or promoted adipogenesis, respectively. In vivo, in response to caloric excess, adipocyte precursor (AP)-specific Tmbim1 knockout (KO) mice displayed WAT hyperplasia and improved systemic metabolic health, while overexpression of Tmbim1 in transgenic mice showed the opposite effects. Moreover, mature adipocyte-specific Tmbim1 KO did not affect WAT cellularity or nutrient homeostasis. Mechanistically, TMBIM1 binds to and promotes the autoubiquitination and degradation of NEDD4, which is an E3 ligase that stabilizes PPARγ. Our data show that TMBIM1 is a potent repressor of adipogenesis and a potential therapeutic target for obesity-related metabolic disease.

[Effect of posterior condylar offset on knee flexion after high-flex posterior-stabilized total knee arthroplasty].

OBJECTIVE: To determine the effect of the posterior condylar offset (PCO) on intra- and post-operative knee flexion after total knee arthroplasty (TKA) using a high-flex posterior-stabilized (PS) fixed-bearing prosthesis and to discuss it's importance in femoral prosthesis design. METHODS: The clinical and radiographic materials of 100 consecutive patients (100 knees) were prospectively studied, including 50 men and 50 women, who had undergone primary NexGen LPS-Flex TKAs for end-stage osteoarthritis. All operations were performed by a single surgeon using the same operative technique between March 2005 and October 2006. Pearson's regression analysis was used to assess the relationship between the difference in the pre- and post-operative PCO on true lateral radiographs and the change in knee range of flexion (ROF) under non-weight-bearing conditions. RESULTS: The decrease of the corrected PCO was (3.4 ± 3.3) mm compared with the preoperative value, the restoration of PCO was better in male than female [female (-5.4 ± 3.1) mm vs. male (-1.5 ± 2.0) mm, P < 0.05]. The difference in the corrected PCO after PS TKA demonstrated significantly correlated with the change in 2 years postoperative ROF in male and female, respectively (P < 0.05). While no statistically correlation was observed in the overall group (P > 0.05). Intraoperatively, the difference in the corrected PCO was significantly correlated with the change in ROF in male, female, and the overall group, respectively (P < 0.05). CONCLUSIONS: Restoration of PCO plays an important role in the optimization of knee flexion after high-flex PS TKA. Femoral components based on Caucasian anatomic characteristics could not match the native anatomy of distal femurs of Chinese population especially female Chinese. Rotated resection of distal femur with anterior referencing technique usually leads to a decreased PCO and therefore reduces maximal obtainable flexion. Sexual dimorphism in humans and anatomic variations in various ethnic groups should be seriously considered in total knee prosthesis design.

Reduced Lipocalin 2 Expression Contributes to Vincristine Resistance in Human Colon Cancer Cells.

BACKGROUND: Vincristine (VCR) resistance can lead to cancer chemotherapy failure. Although changes in gene expression are responsible for drug resistance, the specific identities and roles of these genes remain unclear. OBJECTIVE: In this study, we aimed to identify differentially expressed genes and mechanisms of VCR resistance in colorectal cancer (CRC) cells. METHODS: A VCR-resistant CRC cell line (HCT-8/VCR) was established, and differentially expressed proteins between HCT-8 and HCT-8/VCR cells were screened using a human cytokine array; the results were confirmed by reverse transcription polymerase chain reaction and Western blotting. Furthermore, differentially expressed proteins were downregulated using siRNA, and cell proliferation and apoptosis were assessed by Cell Counting Kit-8 assay and flow cytometry, respectively. RESULTS: Compared with HCT-8 CRC cells, HCT-8/VCR cells showed downregulation of lipocalin 2 (LCN2). We found that siRNA-mediated downregulation of LCN2 in HCT-8 cells significantly increased VCR resistance. Furthermore, when we downregulated LCN2, we observed significant decreases in apoptosis, but no significant effect on cell cycle. CONCLUSION: Overall, these results demonstrate that LCN2 plays an important role in VCR resistance and is a potential therapeutic target for this disease.

Exploring the key genes and pathways of side population cells in human osteosarcoma using gene expression array analysis.

BACKGROUND: Human osteosarcoma (OS) is one of the most common primary bone sarcoma, because of early metastasis and few treatment strategies. It has been reported that the tumorigenicity and self-renewal capacity of side population (SP) cells play roles in human OS via regulating of target genes. This study aims to complement the differentially expressed genes (DEGs) that regulated between the SP cells and the non-SP cells from primary human OS and identify their functions and molecular pathways associated with OS. METHODS: The gene expression profile GSE63390 was downloaded, and bioinformatics analysis was made. RESULTS: One hundred forty-one DEGs totally were identified. Among them, 72 DEGs (51.06%) were overexpressed, and the remaining 69 DEGs (48.94%) were underexpressed. Gene ontology (GO) and pathway enrichment analysis of target genes were performed. We furthermore identified some relevant core genes using gene-gene interaction network analysis such as EIF4E, FAU, HSPD1, IL-6, and KISS1, which may have a relationship with the development process of OS. We also discovered that EIF4E/mTOR signaling pathway could be a potential research target for therapy and tumorigenesis of OS. CONCLUSION: This analysis provides a comprehensive understanding of the roles of DEGs coming from SP cells in the development of OS. However, these predictions need further experimental validation in future studies.

[Treatment of valgus deformity by total knee arthroplasty with modified Ranawat soft tissue balance technique].

OBJECTIVE: To evaluate the efficacy of modified Ranawat soft tissue balance technique on total knee arthroplasty (TKA). METHODS: From January 2004 to June 2008, 34 cases (44 knees) of valgus deformity were treated with TKA. There were 5 males (5 knees) and 29 females (39 knees), aged 55-79 years old (average 60.3 years old) and including 18 left knees and 26 right knees. The deformity was caused by osteoarthritis in 9 cases, by rheumatoid arthritis in 19 cases, and by traumatic arthritis in 6 cases. According to Ranawat classification, there were 5 cases (5 knees) of type I and 29 cases (39 knees) of type II. All patients were performed modified Ranawat soft tissue balance technology. RESULTS: The operative time was (65 +/- 7) minutes. Burst fracture of femoral condyle occurred and internal fixation was selected in 1 case of rheumatoid arthritis. Small incision necrosis occurred and healed after debridement in 1 case of rheumatoid arthritis. Incision healed by first intention in other cases. Adhesions occurred in 1 case (1 knee) and hydrarthrosis in 4 cases (4 knees), all cured after symptomatic treatment. All patients were followed up 6 months to 5 years with an average of 2.6 years. All patients had no complications of deep vein thrombosis, dislocation, vascular injury and nerve injury. X-ray films showed no signs of prosthesis loosening and infection at 1 year after operation. The X-ray films showed statistically significant differences (P < 0.05) in anatomic valgus angulation between preoperation and 1 week after operation [(25.4 +/- 3.1) degrees vs (3.8 +/- 1.2) degrees]. There were statistically significant differences in modified KSS score between preoperation and 1, 2 years postoperatively (P < 0.05). CONCLUSION: It is a simple and effective way to treat the valgus deformity with modified Ranawat soft tissue balance technique in TKA, which can achieve the satisfactory results in the knee stability, the range of motion and the deformity correction.

[An analysis of operation-correlated complications of total knee arthroplasty].

OBJECTIVE: To summarize and analyze the operation-correlated complications of total knee arthroplasty (TKA) with posterior stabilized prosthesis (PS). METHODS: From October 2000 to October 2007, 707 cases (816 knees) underwent TKA. All the TKA knees were divided equally into former and latter groups according to the operation time (408 knees for each group). In the former group (October 2000 to January 2005, n=350), there were 63 males (84 knees) and 287 females (324 knees), aging (63.5 +/- 7.8) years. A total of 198 left knees affected and 210 right knees affected; 292 single knees affected and 58 both knees affected. There were 304 knees of osteoarthritis, 84 knees of rheumatoid arthritis and 20 knees of other disease. The disease course was (9.3 +/- 5.6) years. The knee range of motion (ROM) was (97.2 +/- 8.7) degrees, the HSS score was 47.4 +/- 12.2. In the latter group (January 2005 to October 2007, n=357), there were 77 males (92 knees) and 280 females (316 knees), aging (62.7 +/- 6.3) years. A total of 221 left knees affected and 187 right knees affected; 306 single knees affected and 51 both knees affected. There were 278 knees of osteoarthritis, 109 knees of rheumatoid arthritis and 21 knees of other disease. The disease course was (8.6 +/- 5.1) years. The knee ROM was (101.1 +/- 10.3) degrees, the HSS score was 49.3 +/- 11.2. We modified the method of lateral patellar retinaculum release and used a new femoral rotational alignment technique in the latter group. The intraoperative and postoperative complications were divided into mild, moderate and severe. The HSS score, ROM and complications of the knee were compared and analyzed statistically during the follow-up. RESULTS: The former group was followed up 5.3 years (1 to 7 years), complications occurred in 278 knees (68.1%), including mild in 136 knees (33.3%), moderate in 135 knees (33.1%) and severe in 7 knees (1.7%). The latter group was followed up 2.1 years (0.5 to 3.5 years), complications occurred in 159 knees (39.0%), including mild in 111 knees (27.2%), moderate in 47 knees (11.5%) and severe in 1 knee (0.2%), and there was significant difference between them (P < 0.001). There was significant difference in increased ROM between the former group (6.0 +/- 3.7) degrees and the latter group (14.4 +/- 4.2) degrees after operation (P < 0.05). There was significant difference in increased HSS score between the former group (36.9 +/- 3.7) and the latter group (44.0 +/- 4.2) after operation (P < 0.05). CONCLUSION: The TKA is a complex operation with innumerable potential complications. To accumulate operative experience and improve surgical skills are the key points to reduce the operation-correlated complications.