Stable Recombinant Cell Lines Research Articles

Preparation of HSV-IgM human-mouse chimeric antibody and development of stable recombinant cell line

In order to achieve large-scale production of HSV-IgM (HSV1, HSV2) human-mouse chimeric antibody in vitro, the gene sequence of the corresponding hybridoma cell was harvested by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) technique to clone the chimeric antibody into eukaryotic expression vectors, and express the target proteins in CHO-S cells. At the same time, the screening process of stable cell lines was optimized, and the pressure conditions of pool construction stage and monoclonal screening stage were explored. Finally, the target protein was purified by protein L affinity purification method and the biological activity was detected. The recombinant IgM antibodies, HSV1 and HSV2, weighted at 899 kDa and 909 kDa respectively, were prepared. The optimal screening pressure was 20P200M (the first phase of pressure) and 50P1000M (the second phase of pressure). The final titer for the monoclonal expression of HSV1-IgM and HSV2-IgM was 1 620 mg/L and 623 mg/L, respectively. This study may facilitate the development of quality control products of HSV1 and HSV2 IgM series recombinant antibodies as well as efficient expression of IgM subtype antibodies in vitro.

Overexpression of glutathione synthetase gene improving redox homeostasis and chicken infectious bursal disease virus propagation in chicken embryo fibroblast DF-1

Infectious bursal disease (IBD) of chickens is an acute, high-contact, lytic infectious disease caused by infectious bursal disease virus (IBDV). The attenuated inactivated vaccine produced by DF-1 cells is an effective control method, but the epidemic protection demands from the world poultry industry remain unfulfilled. To improve the IBDV vaccine production capacity and reduce the economic losses caused by IBDV in chicken, cellular metabolic engineering is performed on host cells. In this study, when analyzing the metabolomic after IBDV infection of DF-1 cells and the exogenous addition of reduced glutathione (GSH), we found that glutathione metabolism had an important role in the propagation of IBDV in DF-1 cells, and the glutathione synthetase gene (gss) could be a limiting regulator in glutathione metabolism. Therefore, three stable recombinant cell lines GSS-L, GSS-M, and GSS-H (gss gene overexpression with low, medium, and high mRNA levels) were screened. We found that the recombinant GSS-M cell line had the optimal regulatory effect with a 7.19 ± 0.93-fold increase in IBDV titer. We performed oxidative stress and redox status analysis on different recombinant cell lines, and found that the overexpression of gss gene significantly enhanced the ability of host cells to resist oxidative stress caused by IBDV infection. This study established a high-efficiency DF-1 cells system for IBDV vaccine production by regulating glutathione metabolism, and underscored the importance of moderate gene expression regulation on the virus reproduction providing a way for rational and precise cell engineering.Graphical

Rational design of transient gene expression process with lipoplexes for high-level therapeutic protein production in HEK293 cells

Abstract Transient gene expression, an attractive technology that enables the rapid production of recombinant proteins for the development of biopharmaceutical candidates at an early stage, offers an interesting alternative to the time-consuming generation of stable recombinant cell lines. In this study, a highly efficient transient gene expression system was established by optimizing significant factors; namely, the host cells, lipid-based transfection reagent, enhancer, and expression vector. HEK293E and Expi293 F cells were evaluated to determine the best host cells to use, and the cell passage number and initial cell density were optimized for high transient gene expression. Cationic liposomes were selected as the vehicle for plasmid DNA delivery and optimized with respect to the co-lipid complex and DNA-to-lipid ratio. Among the cationic lipids, DC-Chol/DOPE gave a more than 2-fold increase in production yield. Following gene delivery into cells, butyric acid (a histone deacetylase inhibitor) supplementation increased protein production by a maximum of 4-folds through transcriptional regulation. Additionally, an expression vector bearing the E77 element at the 3′ end of the polyadenylation signal sequence further enhanced the human-Fc and bevacizumab yields by 7% and 15%, respectively. Finally, our novel combination method was successfully applied to human IgG and Fc protein production using Expi293 F cells.

Accelerating stable recombinant cell line development by targeted integration

Targeted integration (TI) allows fast and reproducible genetic modification of well characterized previously tagged host cells thus generating producer cells with predictable qualities. Concurrently, timelines are cut by 50% compared to random integration (RI) based cell line development. In contrast to commonly low productivities of cell lines generated by TI, we developed a system for CHO cells leading to productivities of more than 1 g/L within weeks using the TurboCell™ platform. The system is based on CHO K1 cells that have been tagged with a GFP expression cassette flanked by recombinase recognition sites. Following GFP based FACS enrichment and cloning of the tagged cells, over 4000 clones were screened for growth, productivity, GFP expression stability and integration status of the GFP expression cassette. The best clones were selected to be used as Master TurboCell (MTC) host cell lines for recombinant cell line development.

Semliki forest virus as a vector: pros and cons for its use in biopharmaceuticals production

The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale.

Establishment of a Chinook salmon cell line with an inducible gene expression system

We have isolated a stable recombinant cell line CHSE-TOF5 derived from the Chinook salmon (Oncorhynchus tshawytscha) embryo cells for use as an inducible expression system. The cells were transfected with the pTet-Off plasmid from the Tet On/Off Clontech system, carrying a G418 resistance gene. Several G418-resistant clones were subcultured and characterised by quantitative PCR (qPCR) and by transient transfection. The level of expression of transcriptional activator was measured by qPCR in a number of isolated clones, and transient transfection with a pTRE2-hyg-LUC plasmid was used to evaluate the inducibility of these clones. A clone was selected for its relative fast cell growth and good level of inducibility. This genetically engineered cell line is a valuable tool for the fish research community especially in research areas investigating the biological function of proteins from fish or fish pathogens.

Development of a cell‐based assay for screening of phosphodiesterase 10A (PDE10A) inhibitors using a stable recombinant HEK‐293 cell line expressing high levels of PDE10A

The cDNA encoding PDE10A (phosphodiesterase 10A) was cloned and a stable recombinant HEK-293 (human embryonic kidney-293) cell line expressing high levels of PDE10A was generated. Transient transfection of pCRE-Luc plasmid, harbouring the luciferase reporter gene under the control of CRE (cAMP-response element)-binding sequence, into the stable recombinant cell line, followed by treatment with PDE10 inhibitor, resulted in a dose-dependent increase in luciferase activity. This method provides a simple and sensitive cell-based assay for screening of PDE10 inhibitors for development of novel therapeutics for the treatment of neurological disorders.

Cloning, stable expression of human phosphodiesterase 7A and development of an assay for screening of PDE7 selective inhibitors

Phosphodiesterases (PDEs) constitute a superfamily of enzymes that plays an important role in signal transduction by catalysing the hydrolysis of cAMP and cGMP. cDNA encoding PDE7A1 subtype was cloned and a stable recombinant HEK 293 cell line expressing high levels of PDE7A1 was generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene into the stable recombinant cell line and subsequent treatment with PDE7 inhibitor, resulted in a dose-dependent increase in luciferase activity. This method provides a simple and sensitive cell-based assay for screening of PDE7 selective inhibitors for the treatment of T cell mediated diseases.

A reporter gene assay for screening of PDE4 subtype selective inhibitors

Phosphodiesterase (PDE) constitutes a superfamily of enzymes that catalyze the hydrolysis of cAMP and cGMP into their corresponding monophosphates and play an important role in diverse physiological functions. The present study provides a process for identifying PDE4 subtypes selective inhibitors using a reporter gene assay. Stable recombinant HEK-293 cell lines expressing high levels of PDE4A4B, PDE4B2A, and PDE4D3 subtypes individually were generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene under the control of cAMP response element (CRE)-binding sequence, into these stable recombinant cell lines followed by treatment with PDE4 inhibitor, resulted in a dose dependent increase in luciferase activity. This methods provide a novel, simple and sensitive assay for high throughput screening of PDE4 subtype selective inhibitors for treatment of asthma and COPD.

Expression of functional recombinant antibody molecules in insect cell expression systems.

Recombinant single-chain variable-fragment molecules (scFv) were constructed from a cell line expressing a monoclonal antibody against African cassava mosaic virus (ACMV) and expressed in Escherichia coli. DNA sequences that encoded the scFv were manipulated to allow scFv expression in insect cell lines. A recombinant baculovirus containing the scFv cDNA was constructed and large amounts of scFv were produced in each of three insect cell lines infected with the baculovirus. However, the scFv were not secreted into the medium by any of the cell lines despite the scFv having been linked to a honeybee melittin leader sequence. The same scFv cDNA construct was introduced into Drosophila DS2 cells and a stable recombinant cell line was obtained that produced scFv that was secreted into the medium. Culture medium containing the scFv was used directly in enzyme-linked immunosorbent assay (ELISA) tests to detect ACMV in plant tissues. Another construct that encoded the Ckappa domain of human IgG was fused to the C-terminus of the scFv that was produced and expressed in Drosophila cells. This scFv derivative also accumulated in the medium and was more active in ELISA than scFv lacking the Ckappa domain.

Induction of recombinant gene expression in stabily transfected cell lines using attenuated vaccinia virus MVA expressing T7 RNA polymerase with a nuclear localisation signal

There are major drawbacks using vaccinia virus (VV) expressing T7 polymerase for eukaryotic expression. VV is infectious for humans and due to cytosolic replication of Poxviridae, transient transfection of T7 promoter containing plasmids is necessary, which varies in efficiency. Several improvements have been introduced to this system to enhance expression of herpes viral glycoproteins. Stably transfected cell lines were generated with an EBV-based episomal plasmid vector which can be pushed to increasing copy numbers under selective pressure. The avirulent vaccine MVA strain was adopted to generate a safe laboratory vector for inserting the bacteriophage T7 RNA polymerase gene with (+) or without (−)a nuclear localisation signal. Constructs were designed for recombination into the VV haemagglutinin gene as recombinants could not be isolated successfully when inserting into the MVA thymidine kinase locus. Both T7 MVA recombinants induced foreign protein expression in transiently transfected cells but only the T7 −/+ MVA induced target protein expression in stably transfected cells. The level of protein expression by this induction mechanism was comparable to, or superior to levels obtained withVV recombinants expressing the gene under control of the VV 11 k IE promoter. The results suggests that the T7 + MVA virus can be used to induce gene expression in stable recombinant cell lines and offers an attractive and safe alternative to other inducible eucaryotic expression systems.