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Beyond the western blot: The advantages of HAP1 cells



Western blot analysis is commonly the first assay performed to validate gene knock-out status on a protein level, but it is well known that there is a large percentage of non-specific commercial antibodies lacking a proper verification process and detecting false positive proteins with similar molecular weights compared to the protein of interest. Even in the event the western blot analysis is working properly, it still does not carry the significance of an assay that can interrogate the functional state of a gene product. Using a commercially made cell line, such as HAP1 knockouts, can be a big time-saver for researchers looking to develop assays that demonstrate these types of functional gene effects.

Scientific discussion

In this regard, we have created a list of HAP1 top peer reviewed scientific articles that provide more insights into complex experimental designs and demonstrate associated/ first proven functional roles of genes within cellular processes/ signaling pathways. We hope this shows how using HAP1 cells can be of great assistance to set-up reliable validation assays and serve as a methodology platform beyond the western blot.

Recently, Horizon’s HAP1 cell lines have expanded to be used in a wide range of research applications such as studies in virology, mitochondria, apoptosis/ autophagy, genome integrity/ functional genomics, drug validation, to name a few. 

The below selected references give partial insights into complex publications that can hopefully serve as inspiration for how to consider using HAP1 KO and wild-type (WT) cells in your next study:

- In the field of virology, researchers often analyze functional roles of associated genes during the course of infection in human HAP1 cells. Inactivation of host genes can lead to an improved viability of the KO (compared to the WT cells), indicating their essentiality for efficient viral replication. Experimental findings are often based on viability, reporter virus1, virus quantification2 and viral time-course3, plaque diameter/ size assays4, or interferon measurements.5,6

- Inactivation of genes, which presumably play a role in the respiratory chain or structural components in mitochondria, gives rise to a better understanding of the cellular powerhouse. After generating of HAP1 KOs, authors measured the effect via respiration measurements by Seahorse7–10, detection of respiratory chain complexes (MS/MS analysis)8,11, several state-of-the-art microscopy techniques,7–10 as well as pulse labelling11 of mitochondrial translation products and cell lysate activity tests of KO/WT cells.12

- In apoptosis/ autophagy associated research, a pooled HAP1 mutagenesis screening experiment led to identification of negative regulators in biosynthesis pathways by labeling cell specific intermediates with subsequent FACS cell sorting and HAP1 KO validation.13 Further HAP1 cells can be used to characterize WT genes in autophagy processes by immuno-staining via autophagy marker (e.g. LC3 puncta).14

- In drug validation studies, performing dose response curves of different cancer relevant inhibitors in WT- and KO HAP1 cell lines, can provide evidences about the cytotoxicity of inhibitors driven by certain gene products.15

- In the area of genome integrity/ functional genomics, HAP1 KOs can shed light on key players participating in the DNA repair mechanism, indicated by chromosomal breaks in comparison to WT control cells.16

HAP1 KO cells can be purchased ready-to-use or as part of a specified custom cell line engineering projects. In case the KO cell lines should be created in the researcher´s laboratory, we offer a broad spectrum of CRSIPR/Cas9 reagents and Horizon´s Technical Support is willing to provide assistance to generate the KO. In case of custom projects our Cell Line Engineering Team is here to offer several services, ranging from the KO design to any screening/ functional assays.

Author: Jan Korte, Ph.D. | Scientific Support Specialist 2

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Learn more 

Is your antibody binding the right target?  – Featured article

HAP1 cell lines - are they the right cell model for you? – Blog article

5 ways to validate and extend your research with knockout cell line – Blog article 

Frequently asked questions for HAP1 knockout cell lines - Blog article

 

Published HAP1 Whole Genome Sequencing Data:

Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).Essletzbichler, P. et al. Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line. Genome Res. 24, 2059–2065 (2014).

 

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References:
  1. Flint, M. et al. A genome-wide CRISPR screen identifies N-acetylglucosamine-1-phosphate transferase as a potential antiviral target for Ebola virus. Nat. Commun. 10, 285 (2019). DOI: 10.1038/s41467-018-08135-4
  2. Lyoo, H. et al. ACBD3 Is an Essential Pan-enterovirus Host Factor That Mediates the Interaction between Viral 3A Protein and Cellular Protein PI4KB. mBio 10, e02742-18, /mbio/10/1/mBio.02742-18.atom (2019). DOI: 10.1128/mBio.02742-18
  3. Moskovskich, A. et al. The transporters SLC35A1 and SLC30A1 play opposite roles in cell survival upon VSV virus infection. Sci Rep 10471 (2019). https://doi.org/10.1038/s41598-019-46952-9
  4. LaFontaine, E. et al. Ribosomal protein RACK1 facilitates efficient translation of poliovirus and other viral IRESs. http://biorxiv.org/lookup/doi/10.1101/659185 (2019) doi:10.1101/659185.
  5. Pokharel, S. M. et al. Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response. Nat. Commun. 10, 1482 (2019). DOI: 10.1038/s41467-019-09453-x
  6. Shen, Q. et al. RanBP2/Nup358 enhances miRNA activity by sumoylating and stabilizing Argonaute 1. http://biorxiv.org/lookup/doi/10.1101/555896 (2019) doi:10.1101/555896.
  7. Kondadi, A. K. et al. Cristae undergo continuous cycles of fusion and fission in a MICOS-dependent manner. EMBO Rep, 21:e49776 (2020). doi: 10.15252/embr.201949776
  8. Małecki, J. M. et al. Human FAM173A is a mitochondrial lysine-specific methyltransferase that targets adenine nucleotide translocase and affects mitochondrial respiration. J. Biol. Chem. 294, 11654–11664 (2019). doi: 10.1074/jbc.RA119.009045
  9. Małecki, J. M. et al. Lysine methylation by the mitochondrial methyltransferase FAM173B optimizes the function of mitochondrial ATP synthase. J. Biol. Chem. 294, 1128–1141 (2019). doi: 10.1074/jbc.RA118.005473
  10. Gioran, A. et al. Multi‐omics identify xanthine as a pro‐survival metabolite for nematodes with mitochondrial dysfunction. EMBO J. 38, (2019). doi: 10.15252/embj.201899558
  11. Sánchez-Caballero, L. et al. A dual function of TMEM70 in OXPHOS: assembly of complexes I and V. http://biorxiv.org/lookup/doi/10.1101/697185 (2019) doi:10.1101/697185.
  12. Yang, Y., Mohammed, F. S., Zhang, N. & Sauve, A. A. Dihydronicotinamide riboside is a potent NAD + concentration enhancer in vitro and in vivo. J. Biol. Chem. 294, 9295–9307 (2019). doi: 10.1074/jbc.RA118.005772
  13. Cao, J. Y. et al. A Genome-wide Haploid Genetic Screen Identifies Regulators of Glutathione Abundance and Ferroptosis Sensitivity. Cell Rep. 26, 1544-1556.e8 (2019). doi: 10.1016/j.celrep.2019.01.043
  14. Agrotis, A., Pengo, N., Burden, J. J. & Ketteler, R. Redundancy of human ATG4 protease isoforms in autophagy and LC3/GABARAP processing revealed in cells. Autophagy 15, 976–997 (2019). doi: 10.1080/15548627.2019.1569925
  15. Hopkins, T. A. et al. PARP1 Trapping by PARP Inhibitors Drives Cytotoxicity in Both Cancer Cells and Healthy Bone Marrow. Mol. Cancer Res. 17, 409–419 (2019). doi: 10.1158/1541-7786
  16. Xing, M. & Oksenych, V. Genetic interaction between DNA repair factors PAXX , XLF, XRCC4 and DNA‐PKcs in human cells. FEBS Open Bio 9, 1315–1326 (2019). doi: 10.1002/2211-5463.12681