Evaluation of the influenza A replicon for transient expression of recombinant proteins in mammalian cells

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells.     

Comparative Analysis of the Recently Discovered hAT Transposon TcBuster in Human Cells

Background

Transposons are useful tools for creating transgenic organisms, insertional mutagenesis, and genome engineering. TcBuster, a novel hAT-family transposon system derived from the red flour beetle Tribolium castaneum, was shown to be highly active in previous studies in insect embryoes.

Methodology/Principal Findings

We tested TcBuster for its activity in human embryonic kidney 293 (HEK-293) cells. Excision footprints obtained from HEK-293 cells contained small insertions and deletions consistent with a hAT-type repair mechanism of hairpin formation and non-homologous end-joining. Genome-wide analysis of 23,417 piggyBac, 30,303 Sleeping Beauty, and 27,985 TcBuster integrations in HEK-293 cells revealed a uniquely different integration pattern when compared to other transposon systems with regards to genomic elements. TcBuster experimental conditions were optimized to assay TcBuster activity in HEK-293 cells by colony assay selection for a neomycin-containing transposon. Increasing transposon plasmid increased the number of colonies, whereas gene transfer activity dependent on codon-optimized transposase plasmid peaked at 100 ng with decreased colonies at the highest doses of transposase DNA. Expression of the related human proteins Buster1, Buster3, and SCAND3 in HEK-293 cells did not result in genomic integration of the TcBuster transposon. TcBuster, Tol2, and piggyBac were compared directly at different ratios of transposon to transposase and found to be approximately comparable while having their own ratio preferences.

Conclusions/Significance

TcBuster was found to be highly active in mammalian HEK-293 cells and represents a promising tool for mammalian genome engineering.     

Delivery of HIV-1 Nef Protein in Mammalian Cells Using Cell Penetrating Peptides as a Candidate Therapeutic Vaccine

The HIV-1 Nef protein expressed early in viral life cycle has been known as a potent candidate for therapeutic vaccine development. Due to different cell barriers, various cell penetrating peptides (CPPs) such as Pep-1 and CADY-2 have been known to deliver biologically active proteins to cytoplasmic compartments via the plasma membrane. In current study, we firstly evaluated the efficiency of lentiviral vector (pCDH-CMV-MCS-EF1-cGFP-T2A-puro) and eukaryotic expression vector (pEGFP-N1) for expression of HIV-1 Nef protein in HEK-293T cells using TurboFect transfection reagent. Our results showed that both vectors can effectively express the Nef proteins within the target cell. The pEGFP-N1 was more effective than pCDH-GFP for protein expression. Furthermore, Nef protein was expressed in E. coli as GST-Nef fusion and transfected by the amphipathic CPPs including Pep-1 and CADY-2 into HEK-293T cells. The size and morphology of the GST-Nef/CPP complexes were evaluated by scanning electron microscopy, and Zetasizer. Our data indicated that the recombinant GST-Nef protein generated in BL21 strain migrated as a clear band of ~50 kDa in SDS-PAGE. The CPP/GST-Nef nanoparticles were formed with a diameter of below 200 nm and notably delivered into HEK-293T cells. Generally, the Nef protein was expressed in prokaryotic and eukaryotic expression systems using different vectors and efficiently transfected in mammalian cells using various delivery systems. The in vitro efficient delivery of HIV-1 Nef gene and also its protein supports the potential of Nef DNA constructs and CPPs as potent carriers of Nef protein for HIV vaccine design in Future.     

Generation of stable cell line by using chitosan as gene delivery system

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.     

Delivery of molecular cargoes in normal and cancer cell lines using non-viral delivery systems

Objective

In this study, transfection efficiency of human papillomavirus (HPV) E7 DNA and protein constructs into HEK-293T normal cell line, and A549 and TC-1 tumor cell lines was evaluated by four delivery systems including supercharge GFP, hPP10 cell penetrating peptide, TurboFect and Lipofectamine using fluorescence microscopy and flow cytometry.

Results

The results indicated that Lipofectamine 2000 and TurboFect produced more effective transfection for GFP and E7-GFP DNA constructs in HEK-293T cells compared to in A549 and TC-1 cells (p?<?0.05). In contrast, the supercharge GFP was efficient for E7 DNA and E7 protein delivery in both normal cell (~?83.94 and ~?77.01% for HEK-293T), and cancer cells (~?71.69 and ~?67.19% for TC-1, and ~?73.86 and ~?67.49% for A549), respectively. Indeed, in these cell lines, transfection efficiency by +36 GFP reached ~?60–80%. Moreover, the hPP10 produced the best transfection result for E7-GFP protein in HEK-293T cells (~?63.66%) compared to TurboFect (~?32.95%); however, the efficiency level of hPP10 was only ~?17.51 and ~?16.36% in TC-1 and A549 cells.

Conclusions

Our data suggested that the supercharge GFP is the most suitable transfection vehicle for DNA and protein delivery into TC-1 and A549 tumor cell lines compared to other carriers.

     

Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.     

Transient gene expression in suspension HEK-293 cells: application to large-scale protein production

Recent advances in genomics, proteomics, and structural biology raised the general need for significant amounts of pure recombinant protein (r-protein). Because of the difficulty in obtaining in some cases proper protein folding in bacteria, several methods have been established to obtain large amounts of r-proteins by transgene expression in mammalian cells. We have developed three nonviral DNA transfer protocols for suspension-adapted HEK-293 and CHO cells: (1) a calcium phosphate based method (Ca-Pi), (2) a calcium-mediated method called Calfection, and (3) a polyethylenimine-based method (PEI). The first two methods have already been scaled up to 14 L and 100 L for HEK-293 cells in bioreactors. The third method, entirely serum-free, has been successfully applied to both suspension-adapted CHO and HEK-293 cells. We describe here the application of this technology to the transient expression in suspension cultivated HEK-293 EBNA cells of some out of more than 20 secreted r-proteins, including antibodies, dimeric proteins, and tagged proteins of various complexity. Most of the proteins were expressed from different plasmid vectors within 5-10 days after the availability of the DNA. Transfections were successfully performed from the small scale (1 mL in 12-well microtiter plates) to the 2 L scale. The results reported made it possible to establish an optimized cell culture and transfection protocol that minimizes batch-to-batch variations in protein expression. The work presented here proves the applicability and robustness of transient transfection technology for the expression of a variety of recombinant proteins.     

The new pLAI (<Emphasis Type="Italic">lux</Emphasis> regulon based auto-inducible) expression system for recombinant protein production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction) prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli.     

High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI

Recombinant proteins are of great commercial and scientific interest. Yet, most production methods in mammalian cells involve the time- and labor-consuming step of creating stable cell lines. Production methods based on transient gene expression are advantageous in terms of speed and versatility; yet, depending on the transfection protocol, transient transfection faces some bottlenecks such as a priori complex formation, limitations in terms of transfection and production media used and the need for medium exchange prior to and/or after transfection. Published protocols for transfection of suspension-adapted HEK-293 cells with polyethyleneimine have shown great promise in overcoming some of these bottlenecks, but still require a priori complex formation for optimal yields and limit the choice of transfection and production media. Here, we report successful in situ transfection of suspension-adapted HEK-293 cells with 25-kDa linear polyethyleneimine at densities up to 20 x 10(6) cells/mL in complex media followed by production at lower cell densities (1 x 10(6) cells/mL). After concentrating cells to such high densities, transfection of HEK-293 cells becomes possible in most commonly used media and is not restricted to a specific medium. Furthermore, there is no need to make transfection complexes a priori, a step that prevents inline sterile filtration of the DNA bulk for transfection, an important consideration when scaling processes up to 100 or 1,000 L. Finally, transfecting HEK-293 cells at high density in complex media is superior to existing transfection protocols and doubles yields of recombinant protein obtainable by transient gene expression.     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.     

Reduced glutamine concentration improves protein production in growth-arrested CHO-DG44 and HEK-293E cells

For most cultivated mammalian cells, glutamine is an essential medium component. However, glutamine consumption results in the production of ammonia, a cytotoxic byproduct. Here we investigated the effect of glutamine reduction on recombinant protein production and ammonia accumulation in transiently transfected CHO and HEK-293E cells maintained under conditions of growth arrest. Maximum transient recombinant protein yields were observed in HEK-293E cultures without glutamine and in CHO cultures with 2 mM glutamine. The initial concentration of glutamine correlated with the level of ammonia accumulation in each culture. For both a stable CHO-derived cell line and a polyclonal population of recombinant CHO cells grown under conditions of mild hypothermia, the highest volumetric protein productivity was observed in cultures without glutamine. Here, the level of ammonia accumulation also corresponded to the initial glutamine concentration. Our data demonstrate that reduction of glutamine in the medium is an effective approach to improve protein production in both transiently and stably transfected mammalian cells when applying conditions that reduce or arrest the growth of these cells.     

A fed‐batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric‐truncated form of tissue plasminogen activator

Aims

A novel chimeric‐truncated form of tissue‐type plasminogen activator (t‐PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed‐batch processes.

Methods and Results

The expression cassette for the novel t‐PA was prepared in pET‐28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase^® Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t‐PA Assay Kit. The fed‐batch fermentation mode, coupled with a Ni‐NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46·66 IU mg^?1) compared to traditional batch mode (35·8 IU mg^?1).

Conclusions

Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed‐batch cultivation methods showed the potential to replace miss‐folded formats of protein with proper folded, soluble form with improved potency.

Significance and Impact of the Study

Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post‐translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.     

Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.     

Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells

Background

Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity.

Results

We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNα2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNα2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNα2b with a recovery greater than 70%. Purified IFNα2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNα2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNα2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNα2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNα2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNα2b produced naturally by lymphocytes. Further, the HEK293-produced IFNα2b is biologically active as shown with reporter gene and antiviral assays.

Conclusion

These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNα2b.     

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

Background

Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use.

Results

We have constructed a Cauliflower mosaic virus (CaMV) 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration) of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV) resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI). The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date.

Conclusion

These modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.     

PKC isoforms interact with and phosphorylate DNMT1

Background

DNA methyltransferase 1 (DNMT1) has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC) may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization.

Results

Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity.

Conclusions

Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.     

Induction of protein body formation in plant leaves by elastin-like polypeptide fusions

Background  

Elastin-like polypeptides are synthetic biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the simple non-chromatographic purification of recombinant proteins. In addition, elastin-like polypeptide fusions have been shown to enhance the accumulation of a range of different recombinant proteins in plants, thus addressing the major limitation of plant-based expression systems, which is a low production yield. This study's main objectives were to determine the general utility of elastin-like polypeptide protein fusions in various intracellular compartments and to elucidate elastin-like polypeptide's mechanism of action for increasing recombinant protein accumulation in the endoplasmic reticulum of plants.     

Enhancing Protein Expression in HEK-293 Cells by Lowering Culture Temperature

Animal cells and cell lines, such as HEK-293 cells, are commonly cultured at 37°C. These cells are often used to express recombinant proteins. Having a higher expression level or a higher protein yield is generally desirable. As we demonstrate in this study, dropping culture temperature to 33°C, but not lower, 24 hours after transient transfection in HEK-293S cells will give rise to ~1.5-fold higher expression of green fluorescent protein (GFP) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. By following the time course of the GFP-expressing cells growing at 37°C and 33°C from 24 hours after transfection (including 19 hours recovery at 37°C in the normal growth medium), we found that a mild hypothermia (i.e., 33°C) reduces the growth rate of HEK-293S cells, while increasing cellular productivity of recombinant proteins. As a result, green cells remain undivided in a longer period of time. Not surprisingly, the property of a recombinant protein expressed in the cells grown at 33°C is unaffected, as shown by the use of AMPA receptors. We further demonstrate with the use of PC12 cells that this method may be especially useful when a recombinant protein is difficult to express using a chemical-based, transient transfection method.     

应用CRISPR/Cas9系统构建MTHFD1基因敲除的HEK-293细胞系

目的:利用成簇的、规律间隔的短回文重复序列/Cas9核酸酶(CRISPR/Cas9)基因编辑技术构建亚甲基四氢叶酸脱氢酶1(methylenetetrahydrofolate dehydrogenase 1, MTHFD1))基因敲除人胚肾(HEK-293)稳定细胞系。方法:利用在线软件筛选出评分最高的3条针对MTHFD1基因的单向导RNA (sg RNA),然后合成sg RNA序列并将其插入到含有GFP标签的质粒中;重组质粒转染HEK-293细胞后通过流式细胞仪分选出已被转入sg RNA的单细胞,通过测序确认单克隆细胞系中MTHFD1的DNA序列突变状态;最后应用实时荧光定量多聚核苷酸链式反应(real-time quantitative Polymerase Chain Reaction, RT-q PCR)和蛋白质印迹(Western blot)方法检测单克隆细胞中MTHFD1的m RNA和蛋白表达水平。结果:重组载体中含有正确的sg RNA序列;测序结果显示该细胞系中MTHFD1基因发生了单个碱基插入突变和6个碱基的缺失突变;RT-qPCR结果显示单克隆细胞系中MTHFD1在m RNA水平显著降低;Western blot检测成功构建MTHFD1蛋白缺失的HEK-293细胞。结论:本研究利用CRISPR/Cas9技术成功构建的MTHFD1敲除HEK-293细胞系。