The most comprehensive introduction of HEK293 collections -- Ubigene

The most comprehensive introduction of HEK293 collections -- Ubigene

HEK293 cell line is one of the most frequently used cell lines. Its application in scientific research is second to HeLa, and its industrial application is second to CHO. However, there is a huge family of HEK293, and he application differences between different derived strains? In this article, we will give introduction of HEK293 series cell lines and their different applications in gene editing.

Introduction of HEK293 and its derived strains

HEK293 (human embryonic kidney 293), is derived from human embryonic kidney cells. Due to the integration of about 4.35 kb adenovirus genome segment on chromosome 19, its cell cycle, phenotype, karyotype changed, and became immortalized. It is widely used to produce proteins, vaccines, anti-cancer reagents, recombinant adenovirus coating, etc.

So far, according to different research and production needs, many subtypes and derived cell lines of HEK293 cells have been established (Figure 1). In this chapter, 293T, 293H, 293F and 293S are mainly introduced.

Figure 1. Family tree of HEK293 cell line

293T cell line

HEK293 cell line stably transfected with SV40 largeT antigen and derived as 293T cell line. 293T cell line is as easy to be transfected as HEK293. The efficiency of transfection with calcium phosphate, PEI or liposome can reach more than 90% ^[1]. The SV40 largeT antigen can achieve the amplification of foreign expression vector, and finally increase the expression amount of target gene. In addition to gene expression and protein production, 293T cells are often used for packaging lentivirus, retrovirus and adenovirus.

293H cell line

This cell line is also derived from HEK293 cell line, which can grow rapidly in serum-free culture system with high transfection efficiency and high protein expression performance. This cell line is different from HEK293 and 293T cell lines. It firmly attaches to the plate and can be used not only for plaque detection, but also for production of recombinant proteins. For example, the recombinant protein VIIIFc fusion protein (rfviifc), a long-acting coagulation factor approved for the treatment of Hemophilia A, is currently in short supply on a regular basis and in violation of production quality, indicating that it is still difficult to manufacture rFVIII. rFVIII is a large and multi-domain glycoprotein with significant post-translational modification. Therefore, a key point for McCue, Justin et al. ^[2] in optimizing the production process of rfviifc is to use a human cell line without adding any ingredients from humans or animals. This shows that 293H cell line has biochemical characteristics conducive to the expression of recombinant protein, such as easy transfection, efficient human protein translation processing and production.

293F cell line

293F cell line is a wild type 293 cell line that can grow rapidly in serum-free culture system, and can express protein efficiently. F refers to Fast-growth. 293F transfected with pCMVSPORT6TAg.Neo plasmid and derived to 293FT. It is usually used in the production of lentivirus. 293FTM cell line were obtained from Flp-InTM T-RExTM 293 cell line by introducing the ecotropic reporter plasmid and the MAPPIT (mammalian protein-protein interaction trap) reporter plasmid, and this cell line is commonly used for protein interaction screening.

293S cell line

293S cell line is a suspension cell line, and S refers to Suspension. This cell line can tolerate low Ca²⁺culture system. 293SG cell line is derived from 293S cell line that induced by methanesulfonic acid, screened by ricin, and introduced with pcDNA6/TR plasmid. 293SG is commonly used for the expression of glycosylated proteins. 293SGGD cell line were obtained by transferring pcDNA3.1-zeo-STendoT plasmid into 293SG cell line, and it is mainly used in glycosylation engineering research.

Application of 293 and 293T cell lines in gene editing

According to the above introduction, you can see that 293 and 293T cells have many basic applications in gene editing. Here we have found some cases of 293 gene editing.

1. Bekier, Michael E. et al. ^[3] used CRISPR/Cas9 technology to knockout two types of Golgi stacking proteins GRASP55, GRASP65 in HeLa and HEK293 cell lines. The characterization of these cell lines showed that the single knockout of GRASP55 or GRASP65 damaged the accumulation of Golgi cisternae, while the double knockout of the two GRASP proteins would decompose the entire Golgi apparatus structure. The decomposition of Golgi apparatus not only accelerates protein transport, but also impairs the accurate glycosylation of proteins and lipids on the cell surface. These KO cell lines provide useful tools for studying the mechanism and biological significance of Golgi structure formation, and may be used to study the pathology of Golgi defects, such as Alzheimer's disease, congenital glycosylation disorder, foot and mouth disease, reoxygenation injury, and a variety of cancers.

2. Yin et al. ^[4], in order to obtain RPA12 shRNA expressing HeLa cell line and mCherry RPA12 expressing HeLa cell line, respectively transfected 293T cells with lentiviral plasmid carrying RPA12 shRNA or mCherry-RPA12 and packaging plasmids pH1 and pH2, and then collected lentivirus particles to transduce HeLa cell line. In addition, Debeb, Bisrat G. et al.^[5] found that 293T cells can be easily cultured and passaged as spheres under the condition of serum-free stem cell culture system. Compared with monolayer cells, cells cultured as three-dimensional spheres (3D) in vitro showed higher ALDH1 and CD4⁴⁺/CD2^4- populations. In addition, 3D spheres generated from 293T cells increased the expression of mesenchymal genes, and microRNAs related to self-renewal and transfer were significantly reduced in 3D spheres. Finally, it is believed that the 3D sphere 293T cells show cancer stem cell phenotype, which is an important research tool for studying the molecular and biological mechanisms of cancer stem cells and testing and developing new targets for cancer treatment.

Figure 2. 293T cells form spheres in suspension culture that exhibit cancer stem cell phenotype. 

A, B) Sphere formation in 293T cells seeded in self renewal promoting suspension culture conditions. Passage of 293T spheres gives rise to higher proportion of secondary spheres. C) Flow cytometric analysis of expression of CD24 and CD44 in 293T cells. Cells were cultured under adherent (2D) or tumor-initiating cells enrichment condition (3D). There is an increase of CD4⁴⁺/CD2^4- population in cells cultured in 3D vs. 2D culture conditions. D) Flow cytometric analysis of ALDH activity showed that the 3D culture condition enriches for ALDH-positive cells compared to the 2D culture of 293T cells.

How to choose 293 and 293T for your research?

HEK293 and 293T cells are the two most commonly used cell lines in the 293 family. Because they have many similarities in gene and phenotype, people often get entangled when choose them for research. So how to choose the best one for your subsequent experiments?

By analyzing the genome sequencing result of the offspring cell lines derived from HEK293, it was found that compared with the parent HEK293, these cell lines’ chromosomes were more unstable, and the copy number increased or lost at multiple locations in the genome. According to the actual cases of Ubigene, 293T is prone to have compound heterozygote during gene editing, which is difficult to validate the exact genotype. Therefore, we believe that HEK293 cell line is more suitable for gene editing experiments such as KO, KI or point mutation. In 293T cell line, due to the transfection of the SV40T antigen, the plasmid containing SV40ori could be significantly amplified in the cell line, thus promoting gene expression. In addition, after transfected with SV40 large T antigen, the cell proliferation was significantly improved compared with HEK293, so 293T cell line is more suitable for plasmid transfection, virus packaging and other expressing experiments.

Reference

[1] Lin, Yao-Cheng, et al. "Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations." Nature communications 5.1 (2014): 1-12.

[2] McCue, Justin, et al. "Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein." Biologicals 43.4 (2015): 213-219.

[3] Bekier, Michael E., et al. "Knockout of the Golgi stacking proteins GRASP55 and GRASP65 impairs Golgi structure and function." Molecular biology of the cell 28.21 (2017): 2833-2842.

[4] Yin, Xiaomei, et al. "RNA polymerase I subunit 12 plays opposite roles in cell proliferation and migration." Biochemical and Biophysical Research Communications 560 (2021): 112-118.

[5] Debeb, Bisrat G., et al. "Characterizing cancer cells with cancer stem cell-like features in 293T human embryonic kidney cells." Molecular cancer 9.1 (2010): 1-12.

[6]  Malm M, Saghaleyni R, Lundqvist M, et al. Evolution from adherent to suspension: systems biology of HEK293 cell line development[J]. Scientific reports, 2020, 10(1): 1-15.