Application and practice of multidimensional teaching method in "Cell Engineering" / 生物工程学报

In order to improve the teaching quality of engineering courses, we introduced a multi-dimensional teaching method into the teaching reform of biology majors in colleges based on the portfolio assessment in the curriculum of Cell Engineering. We reformed the knowledge system, teaching form and implementation scheme of this course. By combining the reform of online teaching, interactive teaching, case teaching and other teaching modes, the students mastered the relevant professional knowledge and the scientific and technological frontier of Cell Engineering. Moreover, their learning interest and enthusiasm, ability of analyzing and solving professional problems related to Cell Engineering also improved. The implementation of teaching reform of this course provides a reference for other similar professional courses in colleges.

Therapeutic cell engineering: designing programmable synthetic genetic circuits in mammalian cells

Cell therapy approaches that employ engineered mammalian cells for on-demand production of therapeutic agents in the patient's body are moving beyond proof-of-concept in translational medicine. The therapeutic cells can be customized to sense user-defined signals, process them, and respond in a programmable and predictable way. In this paper, we introduce the available tools and strategies employed to design therapeutic cells. Then, various approaches to control cell behaviors, including open-loop and closed-loop systems, are discussed. We also highlight therapeutic applications of engineered cells for early diagnosis and treatment of various diseases in the clinic and in experimental disease models. Finally, we consider emerging technologies such as digital devices and their potential for incorporation into future cell-based therapies.

Promoting teaching quality by portfolio assessment in Cell Engineering classroom / 生物工程学报

We introduce the portfolio assessment into the classroom teaching reform in the curriculum of Cell Engineering, a specialty course in bioengineering & biotechnology. We established a complete classroom evaluation system that was divided the classroom assessment system of portfolio into four stages including the preparation stage, training stage, implementation stage and exhibition stage. We also discuss the feasibility and necessity of implementing the portfolio evaluation method in the course of cell engineering, the construction of evaluation system, and the key points and matters needing attention in the implementation process. The classroom reform is very productive, not only the classroom atmosphere has been activated, students' learning initiative and autonomy has been enhanced, but also the students' ability to analyze and solve professional problems related to cell engineering technology has been improved. The implementation of classroom teaching reform of this course can provide reference for other similar professional courses in colleges and universities.

Impact of metabolic enzymes overexpression on transient expression of anti-hLAG3 by CHO cells / 生物工程学报

To enhance recombinant protein production by CHO cells, We compared the impact of overexpression of metabolic enzymes, namely pyruvate carboxylase 2 (PYC2), malate dehydrogenase Ⅱ (MDH2), alanine aminotransferase Ⅰ (ALT1), ornithine transcarbamylase (OTC), carbamoyl phosphate synthetase Ⅰ (CPSⅠ), and metabolism related proteins, namely taurine transporter (TAUT) and Vitreoscilla hemoglobin (VHb), on transient expression of anti-hLAG3 by ExpiCHO-S. Overexpression of these 7 proteins could differentially enhance antibody production. OTC, CPSI, MDH2, and PYC2 overexpression could improve antibody titer by 29.2%, 27.6%, 24.1%, and 20.3%, respectively. Specifically, OTC and MDH2 could obviously improve early-stage antibody production rate and the culture period was shortened by 4 days compared with that of the control. In addition, OTC and MDH2 had little impact on the affinity of anti-hLAG3. In most cases, overexpression of these proteins had little impact on the cell growth of ExpiCHO-S. MDH2 and ALT1 overexpression in H293T cells could also improve antibody production. Overall, overexpression of enzymes involved in cellular metabolism is an effective tool to improve antibody production in transient expression system.

Signaling network-based functional cell design / 生物工程学报

Cellular signaling networks act as the central processor to deal with environmental signals and regulate cell function, and determine cell fate. Using synthetic biology approach to engineer cell signaling networks is crucial for ultimately constructing man-made "cell machines". Cellular signaling networks can encode sophisticated cell information by processing quantitatively signaling dynamics, which enables multi-dimensional regulation of functional sub-circuits. Here, we first review the research progresses on the signaling coding mechanisms; and then elaborate the methodologies and applications of cells signaling engineering; finally, we envision that signaling-based cell engineering are important for the increasingly-complicated next generation synthetic biology.

Development of Genetically Modified Chinese Hamster Ovary Host Cells for the Enhancement of Recombinant Tissue Plasminogen Activator Expression

Background: Chinese hamster ovary (CHO) cells are the most commonly used host system for the expression of high quality recombinant proteins. However, the development of stable, high-yielding CHO cell lines is a major bottleneck in the industrial manufacturing of therapeutic proteins. Therefore, different strategies such as the generation of more efficient expression vectors and establishment of genetically engineered host cells have been employed to increase the efficiency of cell line development. In order to examine the possibility of generating improved CHO host cells, cell line engineering approaches were developed based on ceramide transfer protein (CERT), and X-box binding protein 1s (XBP1s). Methods: CHO cells were transfected with CERT S132A, a mutant variant of CERT which is resistant to phosphorylation, or XBP1s expression plasmids, and then stable cell pools were generated. Transient expression of t-PA was examined in engineered cell pools in comparison to un-modified CHO host cells. Results: Overexpression of CERT S132A led to the enhancement of recombinant tissue plasminogen activator (t-PA) expression in transient expression by 50%. On the other hand, it was observed that the ectopic expression of the XBP1s, did not improve the t-PA expression level. Conclusion: The results obtained in this study indicate successful development of the improved CHO host cells through CERT S132A overexpression.

Advances in improving mammalian cells metabolism for recombinant protein production

Background: The production of recombinant proteins for therapeutic use represents a great impact on the biotechnology industry. In this context, established mammalian cell lines, especially CHO cells, have become a standard system for the production of such proteins. Their ability to properly configure and excrete proteins in functional form is an enormous advantage which should be contrasted with their inherent technological limitations. These cell systems exhibit a metabolic behaviour associated with elevated cell proliferation which involves a high consumption of glucose and glutamine, resulting in the rapid depletion of these nutrients in the medium and the accumulation of ammonium and lactate. Both phenomena contribute to the limitation of cell growth, the triggering of apoptotic processes and the loss of quality of the recombinant protein. Results: In this review, the use of alternative substrates and genetic modifications (host cell engineering) are analyzed as tools to overcome those limitations. In general, the results obtained are promising. However, metabolic and physiological phenomena involved in CHO cells are still barely understood. Thus, most of publications are focused on specific modifications rather than giving a systemic perspective. Conclusions: A deeper insight in the integrated understanding of metabolism and cell mechanisms is required in order to define complementary strategies at these two levels, so providing effective means to control nutrients consumption, reduce by-products and increase process productivity.

Construct eukaryotic expression vector with aFGF gene and transfect muscle satellite cells / 中华显微外科杂志

Objective To construct human acidic fibroblast growth factor (aFGF) recombinant eu karyotic expression vector and transfect it into muscle satellite cells(MSCs) of rat,in purpose of further study the method to set up cell bank.Methods The aFGF gene was cloned from human total RNA which was obtained from human skeletal muscle tissue by RT-PCR method.Human interleukin 2 (IL-2) signal peptide sequence (SPS) was obtained by direct chemosynthesis method.Then aFGF and SPS were fused to obtain SPS-aFGF.Finally,directional cloning SPS-aFGF into pEGFP-N1,the recombinant (pEGFP-N1-SPS-aFGF) was obtained.The recombinant was confirmed by endonuclease digestion and DNA sequencing.MSCs were purified by difference-speed adherence method and were ideontified by immunofluorescence assay.The correct cells were divided into 3 groups:Experimental group (aFGF +N 1),control group (N 1),blank group (blank).All the groups were transfected by Lipofectamine 2000TM Reagent,and pEGFP-N1-SPS-aFGF,pEGFP-N1 were respectively added in experimental group and control group while blank group was added none plasmid.Fluorescence microscope was employed to detect transfection efficiency tendency along with time changes.The expression of target gene was detected by fluorescent quantitation PCR and Western blot.Results (1) The sequencing of pEGFP-N1-SPS-aFGF was completely correct and the outcome of endonuclease was equal to actual ban s-ize.(2)The expression of GFP in transfected cells were observed by fluorescencemicroscope and transfection efficiency reached the peak at 72 h.(3)Real-time fluorescent quantitation PCR proved strong aFGF mRNA expression in transfected cells (the average relative expression of experimental group was 1464.95)with aFGF gene,while it was detected a little in the other groups (the average relative expression of control group was 1.016 and blank group was 1.000) (P ＜ 0.05).Western blot also proved strong expression in Experimental group then the other two groups.Conclusion aFGF eukaryotic expression vector was successfully constructed and transfected into MSCs.This study may be expected to obtain some specific functions cells.

Experimental study on VEGF165 transferred dermal multipotential stem cells / 第三军医大学学报

Objective To obtain skin seed cells which can highly express active vascular endothelial growth factor 165(VEGF165) so as to promote refractory wound healing by gene therapy in combination with cell engineering.Methods After pIRES2-EGFP-hVEGF165 was transfected into dermal mesenchymal stem cells(DMSCs) by lipofectin,the expression of VEGF mRNA was detected by RT-PCR,VEGF protein in the supernatant and in the cells were assayed by enzyme-linked immunosorbent assay(ELISA) and Westen blotting respectively.To evaluate the activity of VEGF secreted by transfected DMSCs,hECV304 was cultured with the supernatant of transfected DMSCs and its proliferation activity was analyzed by MTT assays.Meanwhile,the proliferation activity of VEGF-transfected DMSCs and non transfected DMSCs was investigated by MTT.Results The results of RT-PCR,ELISA and Western blot demonstrated that the expression of VEGF in transfected DMSCs was about 1.6 times than that of control DMSCs.The product not only enhanced the proliferation of hECV304 but also increased the proliferation of transfected DMSCs.Conclusion The plasmid pIRES2-EGFP-hVEGF165 is successfully transfected into DMSCs with the aid of lipotransfection,and hVEGF165-transfected DMSCs might high-efficiently secrete highly active VEGF165.