Angiotensin-converting enzyme 2 (ACE2) is a key protein in the renin-angiotensin system (RAS) that plays a critical role in cardiovascular health and fluid balance. ACE2 is primarily found on the surface of various cell types, including alveolar epithelial cells, small intestinal enterocytes, endothelial cells, and arterial smooth muscle cells. It has several critical physiological functions, including converting angiotensin II, a potent vasoconstrictor, into angiotensin-(1-7), which promotes vasodilation and has anti-inflammatory properties.
ACE2 has received extensive attention during the COVID-19 pandemic because it is a receptor for the SARS-CoV-2 virus. The viral spike protein binds to ACE2 to facilitate entry into host cells. The widespread expression of ACE2 in different tissues explains the multi-organ effects frequently observed in COVID-19 patients. This interaction has prompted extensive research into inhibitors or modulators of ACE2 as potential therapeutic avenues for COVID-19. In addition to COVID-19, ACE2 remains a focus of research in hypertension, heart failure, and kidney disease. Understanding the dynamics of ACE2 within the RAS and its broader impact may lead to new treatments for a variety of cardiovascular and inflammatory diseases. ACE2 therefore remains a protein of great biomedical importance, impacting multiple areas of health and disease.
Case Study 1
Studies have shown that SARS-CoV-2 infects human monocytes, monocyte-derived macrophages, and dendritic cells in vitro, which may play an important role in the pathogenesis of COVID-19. However, whether SARS-CoV-2 can infect lymphocytes that do not express ACE2 and cause lymphocytopenia remains unknown. Here, the study showed that activated T lymphocytes can be infected by SARS-CoV-2 in an ACE2-independent manner. This infection led to significant apoptosis of T cells in vitro or in COVID-19 patients. The results of this study help understand lymphocytopenia caused by SARS-CoV-2 infection.
ACE2 is widely believed to be the entry receptor for SARS-CoV-2. However, the major cell population in peripheral blood cells (PBCs) expressed extremely low levels of ACE2, raising the question of whether ACE2 also mediates SARS-CoV-2 viral entry into T cells. The researchers first tested whether ACE2 knockdown could inhibit SARS-CoV-2 infection of T cells. The data showed that ACE2 was successfully knocked down by ACE2-shRNA in Caco2 cells. Jurkat T cells did not express detectable ACE2 under either mock or knockdown conditions (Figure 1a). Correspondingly, ACE2 knockdown resulted in a significant reduction in SARS-CoV-2 infection in Caco2 cells, but had no such effect in Jurkat T cells (Figure 1b). To further confirm this finding, ACE2 was knocked out in Caco2 and Jurkat cells (Figure 1c). Similar to ACE2 knockdown cells, the viral load decreased in ACE2 knockout cells-Caco2, but not in ACE2 knockout cells-Jurkat (Figure 1d). These results suggest that SARS-CoV-2 infects T cells in an ACE2-independent manner.
Figure 1. SARS-CoV-2 infection of T cell is spike-ACE2/TMPRSS2-independent. (Shen X R, et al., 2022)
Case Study 2
Validation of top-ranked genes using pseudoviral and replication-competent SARS-CoV-2 infection assays.
Paper
2025
Background
Researchers employed a membrane-focused CRISPR activation screen in cells with or without ACE2 to identify determinants of SARS-CoV-2 tissue tropism. They uncovered multiple candidate membrane proteins and validated the endo-lysosomal protease legumain (LGMN) and potassium channel KCNA6 as novel host factors that promote viral entry. Endogenous disruption or pharmacological inhibition reduced infection by pseudotyped and live SARS-CoV-2; LGMN activity and localization were critical. Clinical scRNA-seq data correlated LGMN expression with viral RNA and disease severity, while KCNA6 marked olfactory cells, suggesting targetable mediators of viral spread.
Key Findings
Using CRISPRa screening of ~6,213 membrane protein genes in 293FT cells with and without ACE2, followed by SARS-CoV-2 spike pseudovirus and replication-competent virus validation, the authors identified legumain (LGMN) and KCNA6 as robust facilitators of infection. Knockout of endogenous LGMN or KCNA6 in gastric, kidney, and T-cell lines decreased infection; LGMN effect required catalytic residues H148/C189 and proper endosomal/cell-surface localization. KCNA6 overexpression promoted spike-mediated entry independently of other KCNA family members, and inhibitors RR-11a and 3,4-DAP reduced infection in a spike-dependent manner. Both factors depended on ACE2 yet did not alter expression of known host factors. Single-cell transcriptomic data showed LGMN in BALF macrophages correlated positively with SARS-CoV-2 RNA and disease severity; KCNA6 was expressed in olfactory OLIG2+ cells. These findings reveal druggable tissue-specific determinants and demonstrate value of membrane-wide CRISPRa screening.
Contribution
Creative Biogene supplied ACE2 KO 293T cells, which were essential for experiments showing LGMN and KCNA6 depend on ACE2 expression to promote SARS-CoV-2 entry.
Case Study 3
Humoral Responses Following XBB.1.5 Booster in Rhesus Macaques.
Paper
2023
Background
This study evaluated a monovalent recombinant spike protein COVID-19 vaccine based on SARS-CoV-2 Omicron XBB.1.5 (Novavax). In mice and nonhuman primates, XBB.1.5 immunization induced neutralizing antibodies against XBB.1.5, XBB.1.16, XBB.2.3, EG.5.1, and XBB.1.16.6, elicited Th1-biased CD4+ T-cell responses, and boosted antibody titers in animals primed with monovalent or bivalent vaccines. The monovalent XBB.1.5 vaccine elicited higher neutralizing titers than a bivalent Prototype + XBB.1.5 vaccine. These preclinical data support updating COVID-19 vaccines to a monovalent XBB.1.5 formulation for the 2023–2024 campaign.
Key Findings
The monovalent XBB.1.5 rS vaccine induced higher pseudovirus neutralizing antibody titers against XBB.1.5, XBB.1.16, and XBB.2.3 than a bivalent Prototype + XBB.1.5 vaccine in mice. Primary immunization with XBB.1.5 or XBB.1.16 rS generated cross-neutralizing antibodies against BA.5, XBB.1.5, XBB.1.16, XBB.2.3, EG.5.1, and XBB.1.16.6, whereas Prototype rS yielded lower titers against contemporary variants. In bivalent-primed mice, an XBB.1.5 or XBB.1.16 booster increased neutralizing titers >35-fold; XBB.1.5 boost also induced detectable BA.2.86 neutralizing antibodies. In rhesus macaques primed with Prototype or bivalent vaccine, an XBB.1.5 booster increased neutralizing titers 39.2- to 243.5-fold against XBB.1.5, XBB.1.16, XBB.2.3, FL.1.5.1, and EG.5.1. XBB.1.5 vaccination also elicited Th1-biased CD4+ T-cell responses against XBB subvariants in mice and NHPs, with no reported side effects. These data support a monovalent XBB.1.5 vaccine update.
Contribution
Creative Biogene provided the HEK293T/ACE2 cell line used for pseudovirus neutralization assays. This reagent was essential for measuring neutralizing antibody titers against SARS-CoV-2 XBB variants in mouse and nonhuman primate sera, directly supporting the study's key immunogenicity conclusions.
The Human ACE2 Knockout Cell Line-HEK293T offers numerous applications in biomedical research, particularly in virology, drug discovery, and molecular biology. Below are some primary applications:
Viral Entry Mechanism Investigation: HEK293T ACE2 knockout cells serve as a critical tool for studying the mechanisms of SARS-CoV-2 entry, as ACE2 is the primary receptor for the virus. By comparing knockout cells to wild-type cells, researchers can gain insights into alternative pathways or co-receptors involved in viral infection.
Antiviral Screening: They are used to screen antiviral compounds and neutralizing antibodies targeting different steps in the viral lifecycle, specifically those that may inhibit the interaction between the virus and the ACE2 receptor.
Functional Analysis of ACE2: Using these knockout cell lines, researchers can study the physiological role of ACE2 under normal and pathological conditions, including its role in cardiovascular diseases, hypertension, and other ACE2-related functions.
Vaccine Development: These cell lines can be used for validating the efficacy of vaccines that target the ACE2-SARS-CoV-2 interaction, helping to ensure that vaccine candidates effectively block this critical interaction in a human cell context.
High-Throughput Screening: Utilize the knockout cell line in high-throughput screens to discover novel therapeutic targets and compounds.
Customer Q&As
What is the recommended growth medium? Does it require antibiotic selection?
A: DMEM supplemented with 10% fetal bovine serum. <br> It is not required to add the selection antibiotics when culturing the KO cells.
How is the knockout cell line validated?
A: The knockout cell product is validated by PCR amplification and Sanger Sequencing to confirm the mutation at the genomic level. Please find the detailed mutation info in the datasheet.
Is the product a single clonal cell or mixed cell pool?
A: Single clonal cell.
Can I confirm gene knockout by RT-qPCR?
A: No. This knockout cell product is generated using the CRISPR/Cas9 system to induce small insertions or deletions (indels) resulting in frameshift mutations. Although these frameshift mutations typically disrupt the coding gene, there is a possibility that the non-functional transcript may still be transcribed. Consequently, this could potentially yield misleading results when analyzed by RT-qPCR.
How can I store the cell product?
A: The cell line should be stored in liquid nitrogen for long-term preservation.
Is it possible to get multiple knockout clones for my GOI?
A: For most cases, we often keep at least 2 clones with different frameshift mutations. Please feel free to contact us to check if there are additional available clones.
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Customer Reviews
Fast & Approximate
Human ACE2 Knockout Cell Line - HEK293T is a product that allows for relatively efficient knockout of the human ACE2 gene, and the results can be observed with a variety of assays.
Highly viable
Human ACE2 Knockout Cell Line-HEK293T is critical for our studies on cell signaling pathways. The knockout is effective, and the cells are highly viable and easy to culture.
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